JP4677602B2 - Method for recovering microregions of chromosomes - Google Patents

Description

The present invention relates to a method for recovering a chromosomal microregion, and more particularly, to a method for recovering a chromosomal microregion accurately and efficiently using an atomic force microscope (AFM).
The present invention also relates to a method for determining the base sequence of a chromosome and creating a physical map using DNA as a constituent substance contained in a microregion of the chromosome collected by the above method.

In the conventional genome analysis technology, the whole genome DNA is extracted from the organism to be analyzed, once fragmented to a size of several kb to several tens of kb, cloned into an appropriate sequencing vector, and then the base sequence of each clone And analyzing the relatively short base sequence (usually several hundred bases) thus obtained, and joining the homologous ends at the ends to restore the base sequence of the entire chromosome.
However, especially in eukaryotes, there are many similar sequences and repeated sequences on the genome, and it is impossible to restore the entire sequence only from the base sequence information of the clone. In many cases, the length is several Mb. The limit was to create contigs (the clones aligned based on the homology of the terminal nucleotide sequences and the restriction enzyme cleavage pattern).

Therefore, the position of the contig on the chromosome is usually determined by using genetic map and physical map data obtained from past research, but it is very long for all operations including mapping. The period, enormous effort and budget were required.
In some cases, for example, for the purpose of isolating and identifying useful genes and pathogenic genes, a base sequence or physical map of only a part of the target chromosome may be required. In principle, it is difficult to obtain a base sequence only at a specific location, and it is necessary to perform a considerable amount of base sequence analysis or use data of a genetic map or physical map. It was necessary.

  The cause of this problem is that the length of DNA that can be decoded at a time is limited to a few hundred bases, and the entire genome must first be fragmented into small pieces. At that time, the position information of each fragment (clone) Because it will be lost. This problem can be solved if the target portion of the chromosome is positioned at the nano level, the portion is divided into minute regions and reliably recovered, and the DNA in each minute region can be extracted or amplified. Since the DNA thus obtained has a known position on the chromosome, the above-mentioned restoration problem does not occur, and it is easy to restore the base sequence of the target portion of the chromosome and to create a physical map. Can be done.

  Therefore, for the purpose of recovering the microscopic region of the chromosome so far, recovery with a glass capillary (Non-patent Document 1), recovery of the target part by burning around the target region with a laser (Laser Digestion: Non-patent) Techniques such as Document 2, Patent Document 1 and Patent Document 2) and recovery by an atomic force microscope (AFM) (Non-Patent Document 3 to Non-Patent Document 5) have been developed.

However, in the cutting and collecting with a capillary, since the size of the capillary is large, only a fragment having a size of micrometer or more can be collected, so that it is difficult to collect a minute fragment, and there is a problem in positioning accuracy.
Further, in laser injection, it is difficult to focus the laser to a micrometer or less, and there is a problem in accuracy, and the surroundings are burned out, so that continuous collection is impossible.

On the other hand, in the method of chromosomal cutting and microregion recovery by AFM, the probe is gradually pushed down while reciprocally scanning the chromosomal cutting target region, and finally the probe is pushed down to the substrate surface for cutting. According to this method, the accuracy and the size of the collection area are improved as compared with the collection method using a capillary or a laser.
However, the conventional AFM digestion and recovery method has a problem in that most of the cleaved chromosome constituents remain on the substrate and the recovery rate is extremely low. Further, even if it is attempted to collect adjacent fragments by AFM, the chromosomal constituent material on the boundary line between the two regions is not completely scraped off, and a residue remains.
Therefore, no example of successful continuous cutting using the AFM technique has been reported so far. Furthermore, there is no example that directly verifies that the constituents of the chromosome are actually recovered after the recovery operation by AFM.

H. -J. Ludecke, G. Senger, U. Claussen, B. Horsthemke, Nature, 338, 348 (1989) Shinji Sugano (S. Hadano), Joh-E. Ikeda, Tissue Culture, 22, 55 (1996) C. Mosher, D.C. Jondle, L. Ambrosio, J.A. Vesenka, E.M. Henderson, Scanning Microsc. , 8, 491 (1994) S. Thalhammer, R.A. W. Stark, S. Muullere, J.A. Wienberg, W. M. Heckl, J.A. Struct. Biol. , 119, 232 (1997) S. Iwabuchi, T. Mori, K. Ogawa, K .; Sato, M .; Saito, Y. Morita, T. Ushiki, E .; Tamiya, Arch. Histol. Cytol. , 65, 473 (2002) JP-A-11-148887 JP 2002-202229 A

As mentioned above, several attempts have been made to cut and recover chromosomal microregions, but none of them are perfect technologies, and it is possible to recover microregions below a micrometer continuously. There is no method at this time.
Therefore, an object of the present invention is to provide a method capable of efficiently recovering a microregion containing a chromosomal constituent substance and accurately recovering a plurality of adjacent microregions.
Another object of the present invention is to provide a method for accurately and simply decoding the base sequence of all or any part of the chromosome and a method for creating a physical map based on the collected microregions of the chromosome. To do.

As a result of intensive studies to achieve the above object, the present inventors can efficiently collect a micro area by performing a collection operation in a specific procedure using an AFM probe. In addition, it has been found that continuous recovery can also be realized. It was also found that DNA and the like, which are constituent materials, can be extracted from the collected microscopic region of the chromosome, and can be used for decoding base sequences and creating physical maps.
The present invention is based on such knowledge.

In the present invention according to claim 1, in collecting a microscopic region of a chromosome using an atomic force microscope (AFM), the AFM probe is moved while being pressed toward the substrate on which the chromosome is fixed. Chromosome characterized in that after attaching a microregion containing a constituent substance in a target chromosome to the probe, the microregion containing the constituent substance of the chromosome is recovered by pulling the probe away from the substrate. A method for recovering a minute region of
The magnitude of the force pressing the AFM probe toward the substrate is 1 μN or more and 50 μN or less, and the moving speed of the probe is 0.1 μm or more and 10 μm or less per second,
Of the plurality of microregions constituting all or any part of the chromosome, the first microregion located at the end of the chromosome or any portion is recovered, and subsequently 10 nm or more from the first microregion. For the second micro area with an interval of 5 μm or less, a recovery operation is performed in which the probe is moved in parallel with the moving direction of the probe in the recovery operation of the first micro area. After repeating up to the minute region located at the other end of the part, the collection operation is performed for the minute region existing between the first minute region and the second minute region, and thereafter the same collection operation is sequentially performed for all the minute regions. By repeating until the region can be recovered, all or an arbitrary part of the chromosome is divided into a plurality of continuous microregions and recovered.
The present invention described in claim 2 is the method for recovering a chromosomal microregion according to claim 1, wherein the recovery operation is performed in a solution.
The present invention according to claim 3 is a method for recovering a chromosomal microregion according to claim 2, wherein the solution used for the recovery operation is water, physiological buffer, ethanol, methanol, or an aqueous solution containing a surfactant. is there.

According to a fourth aspect of the invention, according to the movement of the probe, one of claims 1 to 3 performed on the entire width of the chromosomes in the direction forming an angle of 60 degrees to 120 degrees to the long axis of the chromosome This is a method for recovering the microregion of the chromosome .
The present invention according to claim 5 records the collection position on the chromosome for each collection operation, and removes the micro area attached to the probe in each collection operation from the probe or removes the probe from the AFM. The method for recovering a microregion of a chromosome according to any one of claims 1 to 4, wherein the microregion is recovered and stored as a microregion with positional information on a chromosome according to the recovery position on the chromosome of the microregion. is there.
The present invention according to claim 6 is the base sequence obtained by extracting or amplifying DNA contained in a microregion with positional information on a chromosome recovered and stored by the method according to claim 5, and then determining the base sequence. Is a chromosomal base sequence decoding method characterized in that the base sequences of all or any part of the chromosome are determined by sequentially linking the sequences according to the position information.
In the present invention described in claim 7 , after extracting or amplifying DNA contained in a microregion with positional information on a chromosome recovered and stored by the method of claim 5, a part of the base sequence is determined, Based on the positional information of the micro region, a clone containing a sequence homologous to the base sequence of the micro region is selected from clones included in the genomic DNA library prepared by fragmenting genomic DNA and cloning into a vector. A method of preparing a physical map of chromosomes, wherein the selected clones are aligned.
The present invention according to claim 8 is the method for chromosomal sequencing of a chromosome according to claim 6 , characterized in that the chromosome is a chromosome at any one of the period of leptotene, zygotene, pakiten, and diproten. is there.
The present invention according to claim 9 is the method for preparing a physical map of chromosomes according to claim 7 , wherein the chromosome is a chromosome at any one of the leptotene phase, the zygoten phase, the pakiten phase, and the diproten phase. is there.

According to the present invention, a minute region of a chromosome can be efficiently collected, and a continuous collection of a plurality of adjacent minute regions can be performed with high accuracy.
In addition, if the whole or part of the chromosome is divided into minute areas and collected continuously as minute areas with location information, the efficiency of chromosomal sequencing and physical mapping can be dramatically improved compared to conventional methods. It is possible to make it.

Embodiments of the present invention will be described below.
According to the method for recovering a microregion containing a chromosomal component of the present invention, an AFM probe is used to recover the microregion of a chromosome using an atomic force microscope (AFM). By moving while pressing toward the substrate to which the chromosome is fixed, attaching a microregion containing the constituent material in the chromosome to be collected to the probe, by pulling the probe away from the substrate, It is characterized in that a microregion containing a chromosomal component is collected.

In the method of the present invention, the constituent material of the chromosome to be recovered means a material constituting the chromosome, and means a nucleic acid such as DNA, a protein, or the like.
Further, the micro area means an arbitrary part (small part) of the chromosome where the constituent material of the chromosome to be collected exists. The size and position of the minute region need not be particularly limited, and may be freely set according to the intention of a person who desires to collect the minute region.
An atomic force microscope (AFM) is a scanning type that includes a probe at the tip of a cantilever, detects an atomic force acting between the probe and the surface of a sample placed on a substrate, and draws surface irregularities. Any type of probe microscope can be used in the present invention. The size, type, material, and the like of the probe can be selected according to the width of the micro area to be collected.

FIG. 1 shows the principle of the recovery operation in the method of the present invention.
The collection operation in the method of the present invention is performed once so as to traverse or traverse the chromosome 13 to be collected while applying a downward force to the probe 11 (force to press toward the substrate 12 to which the chromosome is fixed). It is moved only in the direction of the left arrow in FIG. 1 and pulled off from the substrate immediately after the movement. Here, pulling means pulling upward with respect to the horizontal plane of the substrate (in the upward arrow direction in FIG. 1), and may be slightly inclined with respect to the horizontal plane of the substrate, and does not necessarily have to be at right angles. .
As a result of such a recovery operation, the microscopic region 14 of the chromosome attached to the probe can be removed and stored without being detached from the probe 11 or detached from the probe.
Therefore, the conventional chromosome cutting and microregion recovery technique (see FIG. 5) by AFM is different from the one in which the probe is reciprocated and stepped down.

Here, the recovery operation, as described in claim 1, the magnitude of the force pressing the probe of the AFM in the substrate direction, 10 pN or more 1000μN less, preferably a 1Myuenu～50myuN, and the moving speed of the probe That is, the speed at the time of movement in the minute region of the chromosome (movement in the direction of the left arrow in FIG. 1) is preferably 1 nm to 300 μm per second, preferably 0.1 μm to 10 μm per second.

The recovery operation may be performed in the atmosphere, but may be performed in a solution as described in claim 2 . As will be described later, when continuously recovering a small region of a large chromosome, it is particularly preferable to carry out in a solution. Examples of the solution for performing the recovery operation in the solution include water, buffer solution, oil, alcohol, and general organic solvents. Among these, in particular, as described in claim 3 , water, physiological buffer Liquid, ethanol, methanol, or an aqueous solution containing a surfactant can be used.
Examples of the physiological buffer include phosphate buffer, Tris buffer, carbonate buffer, and Good buffer. In addition, as the surfactant, any of nonionic, cationic, anionic, amphoteric surfactants, and the like can be used. Typical examples include SDS, Triton X-100, Tween 20 can be mentioned. The content ratio of the surfactant in the aqueous solution is preferably 0.1 to 10%.

Furthermore, the direction of movement of the probe in the minute region of the chromosome (the horizontal arrow in the example of FIG. 1) can be adjusted as appropriate. Generally, as described in claim 4 , it can be performed on the entire width of the chromosome in a direction that forms an angle of 60 degrees to 120 degrees with respect to the long axis of the chromosome. For example, it can be moved at right angles (90 degree angle) with respect to the long axis of the chromosome as in the examples described later. On the other hand, when a plurality of minute regions are continuously collected as described later, the moving direction of the probe is shifted from 90 degrees to some extent with respect to the long axis of the chromosome, preferably 60 to 85 degrees and 95 to 120. A direction that forms an angle of degrees is preferable because a part of DNA contained in adjacent minute regions can be overlapped, and base sequence analysis can be performed efficiently.

  According to the method of the present invention, a microregion containing a chromosomal constituent material can be recovered by performing the recovery operation as described above. The collected microregions of chromosomes contain constituents such as DNA and proteins. According to the method of the present invention, a minute region of a chromosome can be recovered at a high recovery rate without destroying constituents contained in the minute region without leaving a remnant on the substrate. Therefore, by extracting or amplifying DNA from a minute region, it is possible to decode a chromosomal base sequence and create a chromosomal physical map, which are much more labor-saving than conventional genomic analysis techniques.

In the method of the present invention, the above-described recovery operation can be performed for only one place in the chromosome, but all of the chromosome or a portion of interest in the chromosome is divided into minute regions, and the recovery operation is performed for each region. By doing so, it is also possible to perform base sequence decoding and chromosome physical map creation for a wide region of the chromosome.
In this case, it is possible to sequentially proceed with the collection operation for a plurality of microregions constituting all or an arbitrary part of the chromosome. In this case, as for the traveling direction (order) of the collection operation, it can be said that the chromosome or any part is moved one by one from one end to the other end, that is, the probe is shifted by the cutting width. In some cases, a certain amount of the chromosomal component may remain unrecovered between adjacent cutting lines.

Therefore, in order to reduce such a fear, as described in claim 1 , among the plurality of microregions constituting all or any part of the chromosome, the first located at the end of the chromosome or any part. The first micro-region collecting operation is performed, and then the probe moving direction in the first micro-region collecting operation for the second micro-region spaced from the first micro-region by 10 nm to 5 μm. The collection operation is performed by moving the probe in parallel, and then the same collection operation is repeated until the minute region located at the other end of the chromosome or any part of the chromosome. It is preferable that the collection operation is performed for the minute region existing in the middle, and thereafter the same collection operation is sequentially repeated until all the minute regions can be collected. By such a method, the target portion of the chromosome can be divided into a plurality of minute regions and completely recovered, and the possibility that the components of the chromosome that are not recovered remain between adjacent cutting lines can be eliminated. .

The procedure in the case of performing continuous collection by the method of the present invention will be described with reference to FIG. 2A shows the middle of the collection, and FIG. 2B shows the end of the collection.
First, as shown in FIG. 2 (a), a recovery operation is performed on the first microregion 201 located closest to the end A of the chromosome or an arbitrary portion. That is, the collection operation is performed according to the principle shown in FIG. 1 with respect to the entire region of the chromosome or the minute region 201 at one end of an arbitrary portion of interest.
Subsequently, the collection operation is performed on the second minute region 202 spaced from the first minute region 201 by 10 nm to 5 μm, preferably 30 nm to 1 μm. That is, the AFM probe is moved from the first minute region 201 to the second minute region 202 of the nearest chromosome at an appropriate interval, and the collecting operation is performed on the minute region 202. At this time, the movement of the probe needs to be in a direction parallel to the moving direction of the probe in the collection operation of the first minute region 201. Note that the interval between the minute regions can be appropriately adjusted within a range of 10 nm to 5 μm by setting conditions such as the shape of the probe to be used, the moving speed, and the applied force.

Hereinafter, the same collection operation is sequentially repeated in the order of the micro regions 203, 204, 205, 206, and 207 up to the micro region 207 located at the terminal B different from the terminal A on the first micro region 21 side.
When the other end of the chromosome is reached in the second micro area, the recovery operation is performed for the micro area existing in the middle, which will be described later, after the recovery operation of the second micro area. In addition, the size of the micro area | region collect | recovered by each operation may be made to correspond, and may mutually differ.

Through a series of operations up to this point, chromosome microregions 201 to 207 are collected at appropriate intervals, and only a microregion existing in the middle of each microregion remains as shown in FIG. (Right side of FIG. 2).
Therefore, the probe is returned to the minute region 208 existing between the first minute region and the second minute region, and the same collecting operation is performed. Then, the collection operation is performed on the minute region 209 spaced from the first minute region 201 by 10 nm to 5 μm. Subsequently, the collection operation is performed on the minute regions 210, 211, 212, and 213. Also in this case, the movement of the probe needs to be in a direction parallel to the movement direction of the probe in the collection operation of the first minute region 201.
The minute area collected by each of the latter collection operations needs to cover the entire intermediate portion of the minute area collected by each of the preceding collection operations. That is, in the example of FIG. 2, it is necessary to sequentially match the minute region 210 with the entire intermediate portion between the minute regions 201 and 202, and the minute region 211 with the entire intermediate portion between the minute regions 202 and 203.
In this way, it is possible to continuously collect a plurality of microregions 201 to 215 constituting all or an arbitrary part of the chromosome.

It can be seen that the constituent material, for example, DNA, contained in the microregion obtained as a result of the continuous recovery is adjacent on the genome to the DNA contained in the microregion adjacent to the microregion. Therefore, it is clear that the DNA contained in each chromosome can be analyzed by rearranging the plurality of microregions obtained by continuous collection according to the collection position on the original chromosome, and joining the DNA contained in each of the microregions. It is.
Therefore, in order to make it possible to efficiently use constituent materials such as DNA contained in each microregion collected continuously, as genetic information of all or any part of the chromosome, as described in claim 5 , Record the collection position on the chromosome for each collection operation, and remove the micro area attached to the probe in each collection operation from the probe, or remove the probe from the AFM, and the micro area on the chromosome According to the collection position, it is preferable to collect and store as a microregion with position information on the chromosome.
Since the position information on the chromosome can be recorded with nano-level accuracy by AFM, this function can be used to make a minute region with position information on the chromosome.

The microregion continuously collected in this way can be used for elucidation of the base sequence of the chromosome. In other words, by extracting or amplifying DNA contained in microregions with positional information on chromosomes that have been collected and stored continuously, the resulting DNA can be cloned and analyzed in sequence, so that the entire nucleotide sequence or the nucleotide sequence of interest can be decoded. It is.
That is, as described in claim 6 , it is obtained by extracting or amplifying DNA contained in a microregion with positional information on a chromosome recovered and stored by the method of claim 5, and then determining its base sequence to obtain By sequentially linking the base sequences according to the respective position information, the base sequence of all or an arbitrary part of the chromosome can be determined, and the base sequence of the chromosome can be decoded.

Furthermore, DNA taken out from each microregion collected continuously can be used to create a physical map. That is, as described in claim 7 , after extracting or amplifying DNA contained in a microregion with positional information on a chromosome recovered and stored by the method of claim 5 , a part of the base sequence is determined. Selecting a clone containing a sequence homologous to the base sequence of the microregion from clones contained in a genomic DNA library prepared by fragmenting the whole genomic DNA and cloning it into a vector, and position information of the microregion A physical map of the chromosome can be created by aligning the selected clones based on the above.
An example of the creation of a physical map of chromosomes is as follows. First, analyze not only all but a part of DNA as a constituent contained in the collected microregions, and based on the nucleotide sequence information, clones containing homologous sequences from BAC libraries or YAC libraries of genomic DNA. Screen and map the clone to where the microregion on the chromosome was located. If this operation is repeated for each microregion collected continuously, it is possible to select a specific chromosome or a clone located in a specific part on the chromosome, and further align, that is, create a physical map. .

  Here, in each recovery operation in the continuous recovery in the method of the present invention, as described above, with respect to the entire width of the chromosome, the probe is in a direction that forms an angle of 60 degrees to 120 degrees with respect to the long axis of the chromosome. In this way, DNA extracted or amplified from the microregion can be connected, and base sequence analysis and physical map creation can be performed efficiently. As shown in FIG. 3, since the chromosome 31 normally includes two sets of DNA 32, when the probe is moved at the specific angle, the boundary line 35 between the micro regions 33 and 34 between the two DNAs 32. As a result of the deviation, an overlapping portion 36 is generated in both a certain minute region 33 and an adjacent minute region 34, and the base sequence analysis can be performed efficiently.

Moreover, it is preferable to use a chromosome in the meiotic phase, for example, a chromosome in the leptotenic phase, zygotenic phase, pakitenic phase, or diprotenic phase as described in claims 8 and 9 , as a chromosome targeted by the method of the present invention. . Since meiotic chromosomes have an elongated structure that is elongated, the amount of DNA contained is smaller than that of normal somatic chromosomes, even at the same cutting width, and the resolution of genome analysis is reduced. It is possible to improve. Further, at this time, the DNA contained in one chromosome becomes 4 sets, which is advantageous for DNA amplification and analysis of the connection portion with the adjacent region.
Hereinafter, the present invention will be specifically described by way of examples.

According to the method of the present invention, the microregion of the chromosome was collected.
In other words, a silicon probe (brand name: NCH-10T, manufacturer: Nanoworld) is used as the probe for AFM (SPA-800P, manufacturer: Seiko Instruments Nanotechnology), and the probe has a fixed chromosome. 1 mm and 2 μm from the end of the chromosome to be collected are scanned once at a width of 150 nm at a moving speed of 1.0 μm per second while pushing toward the substrate, and then the probe is pulled up by pulling it upward from the substrate. It was. The scanning was performed with the magnitude of the force pressing the probe toward the substrate being about 20 μN. The collection operation was performed in the atmosphere.
FIG. 4 shows an AFM observation image of the chromosome after recovering the minute region of the chromosome.

  On the other hand, the chromosome was cut by a conventional method. That is, the AFM probe was gradually pushed down while reciprocatingly scanning on the region to be cut of the chromosome, and the probe was pushed down to the substrate surface for cutting. FIG. 5 shows an AFM observation image of the chromosome after the microregion is cut by the conventional method.

As a result, in the conventional method (FIG. 5), the minute region of the chromosome 51 is surely scraped off by the movement of the AFM probe and the cut line 52 is formed, but the minute region scraped off when the probe is folded back. The constituent material of was removed from the probe and deposited at the end of the cutting line, became a wreckage 53 and remained on the substrate, and could not be recovered.
On the other hand, in the case of the method of the present invention, no debris was observed at the cutting line 41 as shown in FIG.
From this, it was speculated that the minute region of the chromosome was collected by being attached to the probe.

In order to verify whether or not the microregion of the chromosome recovered by the method of the present invention is actually recovered, the chromosome of the microregion was similarly analyzed for the chromosome stained with the fluorescent dye YOYO-1 that binds nonspecifically to DNA. Collection was performed, and the probe was observed with a fluorescence microscope after collection.
As a result, as shown in FIG. 6, fluorescence was detected at the tip 62 of the probe on the AFM cantilever 61, and it was proved that a microscopic region of the chromosome containing DNA was actually recovered. The reproducibility of recovery is very high, and recovery is successful with a probability of 90% or more.
Further, when the tip of the probe was observed with a scanning electron microscope, it was confirmed that the collected substance 72 derived from the microscopic region of the chromosome was attached to the probe tip 71 as shown in FIG.

According to the method of the present invention, continuous collection of chromosomal microregions was performed. The recovery can be performed in the atmosphere as in Example 1, but in this example, it was examined whether recovery could be performed in a solution ( ultra pure water) .
In accordance with the principle of continuous recovery shown in FIG. 2, first, a recovery operation of a first minute region of a chromosome (a linear region having a length of about 700 nm and a width of about 100 nm) is performed. The collection operation was performed on the second minute region (a linear region having a length of about 700 nm and a width of about 100 nm) in parallel with the first collection operation at an interval of 500 nm. Thereafter, a third collection operation was performed on the minute region remaining in the middle of the first minute region and the second minute region (a linear region having a length of about 700 nm and a width of about 500 nm).
FIG. 8 shows an AFM observation image (a) of the chromosome at the time when the first microregion and the second microregion were collected, and an AFM observation image (b) after the third microregion was collected and the continuous collection was terminated. Indicates.

As is clear from FIG. 8A, two cut lines 81 can be observed on the chromosome at the locations where the first microregion and the second microregion are present. In addition, when the first micro area and the second micro area are collected, an intermediate portion 82 of the micro area existing between the two areas remains as it is. On the other hand, after collecting the third microregion, as is apparent from FIG. 8B, a region 83 where no chromosomal component exists was observed in the intermediate portion 82 of FIG. 8A. (See the right figure in FIG. 8 ), it was verified that the micro area of the intermediate portion 82 was surely recovered.
Since the three microregions on the chromosome were successfully recovered in this way, the recovery operation was similarly performed on a plurality of microregions, so that all or any part of the chromosome was converted into microfragments. It is also clear that it can be divided and recovered continuously.
As a result of the same recovery operation in a solution such as water, physiological buffer solution, ethanol, methanol, and an aqueous solution containing a surfactant, it is possible to recover the components of the chromosome in the same manner, such as a large chromosome In some cases, it was more efficient than recovery in the air.

According to the present invention, a minute region of a chromosome can be efficiently collected, and a continuous collection of a plurality of adjacent minute regions can be performed with high accuracy.
In addition, if the whole or part of the chromosome is divided into minute areas and collected continuously as minute areas with location information, the efficiency of chromosomal sequencing and physical mapping can be dramatically improved compared to conventional methods. It is possible to make it.

It is a figure which shows the principle of collection | recovery operation in the method of this invention. It is explanatory drawing of the procedure at the time of completion | finish of (a) collection | recovery in the case of performing continuous collection | recovery by the method of this invention, and (b) collection | recovery. It is explanatory drawing at the time of cut-and-recovering at a predetermined angle with respect to the long axis of a chromosome. The AFM observation image of the chromosome after collect | recovering the microregion of a chromosome by the method of this invention is shown. The AFM observation image of the chromosome after collect | recovering the microregion of a chromosome by the conventional method is shown. The fluorescence microscope image of the chromosomal component adhering to the probe tip is shown. The scanning electron microscope image of the chromosomal component attached to the probe tip is shown. AFM observation image (a) of the chromosome at the time when the first microregion and the second microregion were collected in Example 2, and the AFM observation image (b) after the third microregion was collected and the continuous collection was terminated. ).

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Probe 12 Substrate 13 Chromosome 14 Microregion A Chromosome or terminal B of arbitrary part Chromosome or other terminal 201-213 Microregion 31 Chromosome 32 DNA
33 and 34 Minute region 35 Boundary line 36 Portion overlapping with minute regions 33 and 34 Cutting line 51 Chromosome 52 Cutting line 53 Remnant 61 Cantilever 62 Tip tip 71 Tip tip 72 Chromosome 81 Cutting line 82 Minute Middle part of the region 83 Region where no chromosomal constituents exist

Claims (9)

When recovering a minute region of a chromosome using an atomic force microscope (AFM), the AFM probe is moved toward the substrate to which the chromosome is fixed, and the constituent substances in the chromosome to be recovered are moved. A method for recovering a microscopic region of a chromosome, comprising: attaching a microregion including the microprobe to the probe, and then recovering the microregion containing a chromosomal constituent material by separating the probe from the substrate. And
The magnitude of the force pressing the AFM probe toward the substrate is 1 μN or more and 50 μN or less, and the moving speed of the probe is 0.1 μm or more and 10 μm or less per second,
Of the plurality of microregions constituting all or any part of the chromosome, the first microregion located at the end of the chromosome or any portion is recovered, and subsequently 10 nm or more from the first microregion. For the second micro area with an interval of 5 μm or less, a recovery operation is performed in which the probe is moved in parallel with the moving direction of the probe in the recovery operation of the first micro area. After repeating up to the minute region located at the other end of the part, the collection operation is performed for the minute region existing between the first minute region and the second minute region, and thereafter the same collection operation is sequentially performed for all the minute regions. A method in which all or an arbitrary part of a chromosome is divided into a plurality of continuous microregions and collected by repeating until the region can be collected.
The method for recovering a chromosomal microregion according to claim 1, wherein the recovery operation is performed in a solution. The method for recovering a chromosomal microregion according to claim 2 , wherein the solution used for the recovery operation is water, physiological buffer, ethanol, methanol, or an aqueous solution containing a surfactant. The method for recovering a microregion of a chromosome according to any one of claims 1 to 3 , wherein the probe is moved with respect to the entire width of the chromosome in a direction that forms an angle of 60 degrees to 120 degrees with respect to the long axis of the chromosome. . Record the collection position on the chromosome for each collection operation, and remove the micro area attached to the probe in each collection operation from the probe, or remove the probe from the AFM, and the micro area on the chromosome 5. The method for recovering a microscopic region of a chromosome according to any one of claims 1 to 4, wherein the microregion is recovered and stored as a microregion with positional information on a chromosome according to the recovery position. 6. After extracting or amplifying DNA contained in a microregion with positional information on a chromosome recovered and stored by the method according to claim 5 , the base sequence is determined, and the obtained base sequences are sequentially linked according to the positional information. To determine the base sequence of all or an arbitrary part of the chromosome. After extracting or amplifying DNA contained in a microregion with positional information on a chromosome recovered and stored by the method according to claim 5, a part of the base sequence is determined, and the whole genomic DNA is fragmented and cloned into a vector A clone containing a sequence homologous to the nucleotide sequence of the microregion is selected from the clones contained in the genomic DNA library prepared as described above, and the selected clones are aligned based on the positional information of the microregion. Chromosome physical mapping method characterized by The method for decoding a base sequence of a chromosome according to claim 6 , wherein the chromosome is a chromosome in any one of the leptotene, zygotene, pakiten, and diproten periods. 8. The method for preparing a physical map of chromosomes according to claim 7 , wherein the chromosome is a chromosome at any one of a leptoten phase, a zygoten phase, a pakiten phase, and a diproten phase.

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