JP4581075B2 - Method and apparatus for decoding genome base sequence and method and apparatus for creating genome physical map - Google Patents

Description

  The present invention relates to a method for decoding a genome base sequence or a method for creating a genome physical map.

Conventional procedures for genome sequence decoding and physical map creation that are generally performed are shown below.
When decoding the base sequence, whole genomic DNA is extracted from the tissue of the target organism, fragmented to a length of several kb by restriction enzyme or sonication, etc., cloned into an appropriate vector and pooled. Random clones are taken out from this pool, and their base sequences are sequentially determined. This operation is performed until the total amount of the determined base sequence is several times that of the entire genome. This means that an arbitrary place on the genome is read several times on average. Usually, the amount of base sequence read is about 2 to 10 times that of the entire genome. The determined base sequence of each clone is accumulated in a database, and by comparing the base sequences at the end of each clone with a computer, adjacent clones are found and connected to restore the original chromosome or genome sequence. However, in reality, it is impossible to restore the whole genome sequence by this method due to the presence of unreadable regions due to mistaken linkages due to similar sequences, insufficient number of duplications, or inability to isolate clones. . Therefore, when a region (contig) of a certain length can be restored, the contig is positioned on the genome by referring to a physical map or genetic map created by past research. The entire base sequence can be restored.

When creating a physical map, the entire genomic DNA extracted from the tissue of the target organism is fragmented from about 100 kb to 300 kb, and BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), PAC (P1 derived artificial chromosome), etc. Create a genomic DNA library by cloning into this vector. Next, the clones contained in the library are cleaved with several types of restriction enzymes, and the positions of the cleavage sites on the clones are determined to create a restriction enzyme map. Thereafter, restriction enzyme maps are compared between the clones, and adjacent clones are determined from analysis of the cleavage pattern (fingerprint method). In parallel with this work, the obtained contig is positioned on the chromosome with reference to the genetic map. Using a marker having sequence information, a PCR primer or a hybridization probe is prepared based on the base sequence, and a library having the marker sequence is identified from the library. If one of the clones contained in a contig is positioned on the chromosome in this way, the position of the contig itself on the chromosome will be revealed.
If the above operations are repeated and the contig can be positioned almost in the entire region of the chromosome, the physical map is completed.
Examples of recovering DNA by cleaving chromosomal microfragments include Biochem. Biophys. Res. Comm. 248, 748 (1998) and J. Struct. Biol. 119, 232 (1997). It is recovered from only one place, and no attempt is made to decode and restore the fragment base sequence. In addition, there is no example in which fragments are completely cut and recovered from a chromosome.

JP-A-8-112110 Biochem. Biophys. Res. Comm. 248, 748 (1998) J. Struct. Biol. 119, 232 (1997)

However, in the conventional method, when total genomic DNA is extracted, fragmented and cloned, the positional information is lost, and it is unclear where each clone was located on the original chromosome (or genome). There was a problem of becoming. Therefore, in both cases of decoding the base sequence and creating the physical map, it is essential to restore the data (base sequence or physical position of the clone) and restore it, which requires a lot of labor, budget, and time. It was.
For example, in the case of base sequence decoding, in higher animals and plants, the sequence of more than tens of millions of DNA fragments must be analyzed, and all these must be compared to find and connect adjacent fragments from the base sequence. Don't be. However, this work requires more labor, budget and time than the determination of the base sequence. Moreover, as described above, it is impossible to completely restore the genome base sequence only from the base sequence data, and it is necessary to refer to the genome physical map and genetic map. In this case, a period of several years or more is required.

On the other hand, even in the case of physical map creation, it is necessary to determine neighboring clones by comparing restriction enzyme cleavage patterns for clones contained in the library, and in order to finally locate the contig on the genome The genetic map had to be referenced, and it took several years and labor to complete it.
In addition, when deciphering a base sequence or creating a physical map of a species with little research accumulation, it is necessary to start with the construction of a genetic map of that species. A period of nearly ten years is required.
Therefore, the present invention collects DNA from the chromosome while recording the location with nanometer level accuracy, determines its base sequence, and restores the whole genome base sequence without the need for a genetic map or physical map. It is an object of the present invention to provide a method and apparatus for decoding a genomic base sequence that can be greatly labor-saving. In addition, DNA is collected from a specific position on the chromosome, and a part of the base sequence information is used to position the genomic library clone on the genome. It is an object of the present invention to provide a method and apparatus for generating a physical genome map that does not need to be provided.

That is, the present invention is a method for decoding a eukaryotic genomic base sequence, the step of sequentially cleaving a eukaryotic chromosome into a plurality of microfragments, and the position of the plurality of microfragments on the chromosome. The genome base sequence decoding method includes the step of recording, and the step of individually collecting the plurality of microfragments.
The genomic nucleotide sequence decoding method according to the present invention further comprises a step of extracting DNA from the recovered microfragments, a step of performing PCR using the extracted DNA as a template and using a primer having a random sequence in part or all thereof, A step of collecting and cloning a plurality of the obtained PCR products to obtain base sequence data of all or part of the plurality of PCR products, the base sequence data being converted into base sequences of the respective end portions of the plurality of PCR products; Reconstructing the base sequence of the entire microfragment by joining together based on the above, and aligning the base sequences of the reconstructed microfragment based on the records of the positions of the plurality of microfragments on the chromosome To restore the full length or a part of the base sequence of the chromosome.
In the genome sequence decoding method of the present invention, the chromosome is preferably a mitotic chromosome or a pachytene chromosome.
In the genome base sequence decoding method of the present invention, the plurality of microfragments preferably have a length of 0.5 μm or less, more preferably 1 nm to 0.5 μm with respect to the long axis direction of the chromosome.
In the genomic base sequence decoding method of the present invention, the step of cutting the chromosome is scraped with a probe of an atomic force microscope or a near-field optical probe microscope, scraped with a glass or metal needle or capillary, or Desirably, this is done by laser rupture.
In this case, scraping is performed by scraping linearly to the central position in the direction perpendicular to the long axis of the chromosome, and then shifting the position in the long axis direction by 1/20 or more and 1/2 or less of the scraping width. It is preferable to scrape the remaining half, or to scrape at an angle of 5 ° to 30 ° from the direction perpendicular to the major axis.
In the genome base sequence decoding method of the present invention, the step of recording the positions of the plurality of microfragments is performed using an atomic force microscope, a near-field optical probe microscope, an optical microscope, a transmission electron microscope, or a scanning electron microscope. It is desirable to do so.

The present invention also provides an observation means capable of observing a chromosome with a resolution of 0.5 μm or less, preferably 1 nm to 0.5 μm, and the chromosome into a microfragment having a length of 0.5 μm or less, preferably 1 nm to 0.5 μm. There is provided a genome base sequence decoding apparatus comprising a cutting means for continuously cutting, a collecting means for collecting the microfragment, and a recording means for recording the position of the collected microfragment on the chromosome.
The genome base sequence decoding apparatus according to the present invention further determines an extraction means for extracting DNA from the microfragment, a cloning means for cloning the extracted DNA, and all or part of the base sequence of the cloned DNA. A base sequence determination means may be provided.

The present invention is also a method for preparing a physical map of a eukaryotic genome, the step of sequentially cleaving a eukaryotic chromosome into a plurality of microfragments, and recording the positions of the plurality of microfragments on the chromosome. And a method for preparing the genome physical map comprising the steps of individually collecting the plurality of microfragments.
The genome physical map creation method according to the present invention further comprises a step of extracting DNA from the recovered microfragments, a step of performing PCR using the extracted DNA as a template and a primer having a random sequence in part or all thereof, A step of recovering and cloning the obtained PCR product, determining the base sequence of the PCR product, fragmenting the genomic DNA extracted from the eukaryote and inserting it into an arbitrary cloning vector, And a step of comparing the base sequence of the PCR product with the base sequence of the clone contained in the genomic DNA library and determining the position of the clone contained in the genomic DNA library on the chromosome. May be.
In the genome physical mapping method of the present invention, the chromosome is preferably a mitotic chromosome or a pachytene chromosome.
In the genome physical map creation method of the present invention, the plurality of microfragments preferably have a length of 0.5 μm or less, more preferably 1 nm to 0.5 μm, with respect to the long axis direction of the chromosome.
In the genome physical mapping method of the present invention, the step of cutting the chromosome is scraped with a probe of an atomic force microscope or a near-field optical probe microscope, scraped with a glass or metal needle or capillary, or Desirably, this is done by laser rupture.
In the genome physical map creation method of the present invention, the step of recording the positions of the plurality of microfragments is performed using an atomic force microscope, a near-field optical probe microscope, an optical microscope, a transmission electron microscope, or a scanning electron microscope. It is desirable to do so.
Furthermore, in the method for preparing a genome physical map of the present invention, the cloning vector is preferably YAC, BAC, or PAC.
The step of collating the base sequences can be performed by PCR or hybridization.
Further, the step of collating the base sequence can be performed on a part of clones included in the genomic DNA library, or can be performed by a PCR product using a clone included in the genomic DNA library as a template. .
Further, in the step of determining the position, in place of the clones contained in the genomic DNA library, the known gene derived from the eukaryote and a part thereof or cDNA and EST (expressed sequence tag) or the eukaryote Any DNA amplified by PCR from genomic DNA or any synthetic DNA having a random sequence can be used.
The genome physical mapping method according to the present invention also extracts the DNA from the recovered microfragment instead of recovering and cloning the PCR product obtained as described above and determining the base sequence of the PCR product. Process, using the extracted DNA as a template and performing PCR using primers that have a random sequence in part or in whole, fluorescent labeling or chemistry on the resulting PCR product or a clone created by cloning the PCR product A step of labeling, a step of fragmenting genomic DNA extracted from the eukaryote and inserting it into an arbitrary cloning vector to create a genomic DNA library, a PCR product or the PCR product subjected to the fluorescent labeling or chemical labeling By cloning a clone produced by cloning the product with a clone contained in the genomic DNA library It can be made comprising the step of determining the position on the chromosome of the clones contained in the genomic DNA library.

The present invention further provides an observation means capable of observing a chromosome with a resolution of 0.5 μm or less, preferably 1 nm to 0.5 μm, and the chromosome into a microfragment having a length of 0.5 μm or less, preferably 1 nm to 0.5 μm. Provided is a genome physical map creation device comprising a cutting means for continuously cutting, a collecting means for collecting the microfragment, and a recording means for recording the position of the collected microfragment on the chromosome.
The genome physical map creation device according to the present invention further comprises an extraction means for extracting DNA from the microfragment, an amplification means for amplifying the extracted DNA, and a position determination means for determining the position of the base sequence of the amplified DNA on the chromosome May be provided.
Hereinafter, embodiments of the present invention will be described.

(I) Genome base sequencing method In the case of genome sequencing, a pachytene chromosome is prepared from a target organism, and chromosomes are sequentially cut at intervals of several tens to 100 nm from the end to collect fragments. At this time, the location of the fragment is recorded.
DNA is extracted from the fragments, amplified by PCR, cloned into a sequencing vector and pooled. From this pool, randomly select clones and determine their nucleotide sequences. This operation is continued until the total base length determined is about 5 times the DNA (50 to 100 kb) contained in the fragment. In conventional genome base sequence decoding, in order to accurately restore the original sequence, it has been necessary to determine a base sequence that is about 10 times, but in the present invention, the range to be restored can be narrowed ( Since the whole genome is one-tenth of a thousand), it is sufficient that the overlap is about 4 to 5 times. The base sequences of the clones are compared, and clones with overlapping ends are found and connected sequentially to restore the base sequences of the fragments. In the present invention, since the position of each fragment on the genome is recorded at the time of cutting, the sequence of the chromosome can be restored by arranging the base sequences in order according to the information.
If desired, in order to efficiently join the boundaries between the fragments and the base sequences of the fragments, it is possible to take a method such as shifting the cutting slightly at the center of the chromosome or cutting it diagonally with a slight angle in the direction of the long axis of the chromosome. . Since the pachytene chromosomes are paired with homologous chromosomes, each chromosome contains 4 sets of genomic DNA. Therefore, an overlapping part can be produced between adjacent fragments by shifting the cutting line or making it oblique.
Since the present invention does not require a genetic map or a physical map and can greatly reduce the sequence restoration work, the genome base sequence decoding period can be dramatically shortened as compared with the conventional method. Furthermore, when analyzing animals and plants that are useful in agriculture, forestry and fisheries for the purpose of isolating useful genes and improving varieties, it is not always necessary to analyze the entire genome. Once the genomic DNA is fragmented, it is impossible to select the DNA from the target location, and partial analysis is difficult. However, in the present invention, if the target portion on the chromosome is specified, the portion may be cut and recovered to decode the base sequence, and the partial analysis can be easily performed. In addition, in the conventional method, clones could not be isolated and an undecipherable region (gap) always occurred. Furthermore, in the conventional method, since operations are performed on the whole genomic DNA, PCR and DNA extraction must be under the same conditions at all locations. On the other hand, according to the method of the present invention, PCR conditions and DNA extraction conditions can be optimally set for each fragment, so that there is a high possibility that DNA can be recovered and the base sequence can be decoded.

(II) Genome physical map creation method When creating a genome physical map, as a preparatory step, the whole genome DNA is extracted from the tissue of the target organism in the same manner as in the conventional method, fragmented to a length of about 100 kb, Create a genomic DNA library by cloning into a vector such as BAC (bacterial artificial chromosome).
Next, a pachytene phase chromosome is prepared, the chromosome is successively cut at intervals of several tens to 100 nm in order from the end, its location is recorded, and the fragment is recovered.
Furthermore, DNA is extracted and amplified by PCR. PCR products are cloned into a vector and pooled, and an arbitrarily small number (several to dozens) of clones are selected from them and their nucleotide sequences are determined. Unlike the case of base sequence decoding, it is not necessary to determine the base sequence of many clones and restore the entire sequence of the fragment. From the BAC clones of the genomic library prepared above, those having the base sequence derived from this fragment are selected by screening or hybridization using the obtained base sequence information (screening). This means that the screened BAC clone was located at the location of the fragment on the chromosome. This operation is performed for all the chromosomal fragments that have been continuously cut, and if the BAC clones can be positioned evenly on the genome, the physical map is completed. In some cases, without PCR, the fragment-derived PCR product or its clone is fluorescently labeled or chemically labeled (for example, biotin can be used) as a probe and hybridized with the BAC clone. By performing this, a BAC clone that matches the fragment can be screened.
The present invention does not require a genetic map, and can be expected to greatly reduce the time and labor required for creating a physical map as compared with the conventional method.
Embodiments of the present invention will be described below with reference to the drawings.

First, an embodiment of the genome sequence decoding method according to the present invention will be described.
FIG. 1 is a flowchart showing a procedure of a method for decoding a genomic base sequence according to the present invention.
First, in step 1, a pachytene chromosome is prepared from an organism to be subjected to genome analysis and fixed on a substrate. The pachytene phase chromosome has an extended structure, and a length of 1 nm in the major axis direction corresponds to a base length of about 1 kb. Therefore, it is a material suitable for increasing the resolution of the position information when cutting and collecting microfragments. However, a normal mitotic chromosome may be used when the positioning of the cutting apparatus and the micro-cutting capability are sufficiently high and fragment cutting and recovery with a width of about 10 nm are possible. Since the height of the pachytene chromosome is several tens of nanometers, the substrate needs to be flat with a maximum surface roughness of several nanometers. Usually, a glass substrate such as a slide glass is used, but a silicon substrate or a resin substrate may be used.
In step 2, the chromosome sample is washed with an acetic acid solution or treated with an enzyme solution (such as protease or RNase). This is because the surface of the chromosome sample prepared by the usual method is covered with impurities. Although these contaminants are extremely thin and transparent, they do not hinder the observation with a microscope. However, when cutting and recovering high-precision chromosome microfragments, the cutting probe causes adsorption or accurate positioning is not possible. There is a problem of not being able to. Therefore, it is desirable to completely remove contaminants by such treatment and to prepare a chromosome sample having a clean surface.

In step 3, chromosomes are sequentially cut sequentially from the end at intervals of several tens to 100 nm, for example, at intervals of 50 to 100 nm, and fragments are collected. At this time, the location of the fragment is recorded. Although it is considered necessary to specify the chromosome to be analyzed, this can usually be determined from the shape of the chromosome (length, ratio of long arm to short arm, etc.). Depending on the species, the size of the chromosomes is almost the same and the shape may be difficult. In such a case, discrimination is made by means such as FISH using a chromosome-specific marker.
In step 4, DNA is extracted from the recovered fragment, amplified by PCR using a primer having all or part of a random sequence, and the product is cloned.
FIG. 2 shows an example in which a region having a width of about 100 nm of a metaphase chromosome is cut and collected, DNA is actually extracted and PCR is performed. In conventional base sequence decoding methods, there was no other method than processing the whole genome under the same conditions, so there was a region where PCR did not occur or clones could not be isolated due to base composition etc. In the invention, since optimum conditions can be determined for each fragment, there is little possibility of failure in PCR or cloning.
In step 5, the obtained clones are randomly selected and the nucleotide sequence is determined. This operation is continued until the determined length of the base sequence becomes 4,5 times the DNA amount of the fragment (usually 50 to 100 kb). This means that all points on the fragment are read on average 4 or 5 times. This number of times is small compared to the case where high-quality data is obtained by the conventional whole genome shotgun method or the like, but since the analysis target region is much smaller than the whole genome, the same or higher accuracy can be obtained.
In step 6, the base sequence data obtained in step 5 is analyzed and connected to restore the entire fragment sequence. If the base sequence is not connected and it is difficult to restore the fragment, the entire region in the fragment may not be uniformly amplified, so the process returns to step 4 to examine the PCR conditions. In addition, because there is a high possibility that a discontinuous portion is included in the adjacent fragment due to the higher order structure of the chromosome, a comparative analysis with the base sequence of the clone derived from the adjacent fragment is also performed. Start decoding from the fragment at one end of the chromosome and finish decoding to the fragment at the other end. If the base sequence of each fragment is arranged in order according to the position information of the fragment recorded in Step 7, the sequence of the chromosome is restored. Is done. In order to accurately connect the base sequences of fragments, it is more efficient that the sequences at both ends of adjacent fragments are slightly overlapped. Therefore, depending on the case, when a minute fragment is cut, a method such as shifting to a key character with a slight shift at the center of the chromosome or cutting at an angle in the direction of the long axis of the chromosome is used. Since the Pakiten chromosomes are paired with homologous chromosomes, one chromosome contains 4 sets of genomic DNA. Therefore, by shifting the cutting line or making it oblique, the cutting position of each set is shifted little by little, and an overlapping portion can be produced between adjacent fragments.

Although the base decoding of one chromosome is completed as described above, this operation may be repeated for all other chromosomes in order to decode the entire genome.
Also, in the agriculture, forestry and fisheries industry, there are cases where it is desired to analyze only a part of a specific chromosome, for example, a region around a useful gene for the purpose of breed improvement. Conventionally, such partial analysis of chromosomes has been difficult to realize, but if the present invention is applied, the target chromosome portion is identified by FISH method or the like, and only that portion is cut and recovered to obtain the base sequence. Analysis can be easily performed by decoding.

Next, an embodiment of the genome physical map creation method according to the present invention will be described.
FIG. 3 is a flowchart showing the procedure.
Referring to FIG. 3, first, in step 1, the whole genomic DNA is extracted from the tissue of the target organism by the same method as in the prior art, and cloned into an appropriate vector to create a library. Here, an example using BAC is shown, but any other vector that can be used generally, such as other YACs and PACs, may be used.
In steps 2 to 5, preparation of the pachytene chromosome, washing, cutting and collection of chromosome fragments, DNA extraction, PCR amplification and cloning are performed.

First, in step 2, a pachytene chromosome is prepared from an organism to be subjected to genome analysis, and is fixed to a substrate. The pachytene phase chromosome has an extended structure, and a length of 1 nm in the major axis direction corresponds to a base length of about 1 kb. Therefore, it is a material suitable for increasing the resolution of the position information when cutting and collecting microfragments. However, a normal mitotic chromosome may be used when the positioning of the cutting apparatus and the micro-cutting capability are sufficiently high and fragment cutting and recovery with a width of about 10 nm are possible. Since the height of the pachytene chromosome is several tens of nanometers, the substrate needs to be flat with a maximum surface roughness of several nanometers. Usually, a glass substrate such as a slide glass is used, but a silicon substrate or a resin substrate may be used.
In step 3, the chromosome sample is washed with an acetic acid solution or treated with an enzyme solution (such as protease or RNase). This is because the surface of the chromosome sample prepared by the usual method is covered with impurities. Although these contaminants are extremely thin and transparent, they do not hinder the observation with a microscope. However, when cutting and recovering high-precision chromosome microfragments, the cutting probe causes adsorption or accurate positioning is not possible. There is a problem of not being able to. Therefore, it is desirable to completely remove contaminants by such treatment and to prepare a chromosome sample having a clean surface.

In step 4, the chromosome is sequentially cut sequentially from the end at intervals of several tens to 100 nm, for example, at intervals of 50 to 100 nm, and fragments are collected. At this time, the location of the fragment is recorded. Although it is considered necessary to specify the chromosome to be analyzed, this can usually be determined from the shape of the chromosome (length, ratio of long arm to short arm, etc.). Depending on the species, the size of the chromosomes is almost the same and the shape may be difficult. In such a case, discrimination is made by means such as FISH using a chromosome-specific marker.
In step 5, DNA is extracted from the recovered fragment, amplified by PCR using a primer having all or part of a random sequence, and the product is cloned.

  In step 6, a small number of arbitrary clones are selected from the clones obtained in step 5, and the base sequence is determined. In step 7, a PCR primer is prepared based on the determined base sequence, and a PCR primer containing the sequence is screened from BAC clones contained in the library by PCR. This means that the screened BAC clone was mapped to the position where the fragment was present on the chromosome (step 8). Since the size of the chromosomal fragment is assumed to be about 100 kb and the insert length of the BAC clone is similar, one BAC clone may be mapped to two adjacent fragment positions. BAC clones that connect fragments can be identified, which is rather useful for physical mapping. In some cases, in step 7, the screening of the BAC clone is not performed by PCR, but a probe based on the base sequence of the clone can be prepared and determined by hybridization. If a membrane or DNA chip with BAC clones blotted at high density is available, this method may be easier to screen. Further, as shown in Step 6 ′, the PCR may be performed by hybridization as described above using the PCR product or clone itself or a DNA fragment derived therefrom as a probe without determining the base sequence. If the above operation is performed from one end of the chromosome to the other end, the physical map of the chromosome is completed. In order to create a physical map of the entire genome, the same operation may be performed for all chromosomes as in the case of base sequence decoding.

FIG. 4 is a schematic diagram showing an outline of one embodiment of the method according to the present invention.
First, as a preparation, using existing methods, genomic DNA is fragmented and cloned into vectors such as YAC and BAC, and a pooled library is created (lower left box, corresponding to step 1 in FIG. 3). ). Micro-fragments are sequentially cut from the chromosomes fixed on the substrate (upper center, corresponding to steps 2 to 4 in FIG. 3), and all or a part of DNA is recovered from each of them and amplified as necessary ( Corresponding to step 5 in FIG. 3). Next, the collected DNA and the library clone are verified. There are the following two means for performing this collation, and either one of them may be performed, or both may be performed to improve accuracy.
Mean 1: All the clones of the library are arrayed and fixed on a chip or membrane, and then recovered from the fragments, or then amplified and subjected to hybridization using a fluorescently or chemically labeled DNA as a probe. . The presence or absence of hybridization is detected by fluorescence or color reaction. It means that the clone that gave a positive reaction has the same base sequence as the fragment-derived DNA (arrow corresponding to “hybridization” in FIG. 4, corresponding to step 6 ′ in FIG. 3). For example, a library It is determined that the clone numbered 1 to 5 was located at the location of fragments 1 to 5 on the chromosome. If the position of the clone is described on the chromosome map based on the position of the fragment recorded in advance, a physical map is created (right end in FIG. 4).
Mean 2: A part of the base sequence of DNA recovered from the fragment or amplified thereafter is decoded (corresponding to step 6 in FIG. 3). PCR primers are synthesized based on the obtained sequence, and PCR is performed on all the library clones to verify whether amplification occurs. It means that the clone obtained by amplification and obtaining the band pattern has the same base sequence as the fragment-derived DNA (arrow corresponding to “PCR, sequence decoding” in FIG. 4, corresponding to step 7 in FIG. 3). Thereafter, a physical map is created in the same manner as the means 1 (right end in FIG. 4).
When creating a physical map, generally hundreds to thousands of markers are positioned on the chromosome. However, in the case of FIG. 4, library clones are used as markers. Hundreds to thousands of library clones are located.

Next, examples of the genome base sequence decoding apparatus and physical map creation apparatus of the present invention will be described.
FIG. 5 shows an example of a genome base sequence decoding and physical map creation device that combines AFM, SNOM / AFM, sample positioning mechanism, scraping mechanism, data recording device, DNA extraction / amplification means, base sequence determination means, and data processing mechanism. Is shown.
In FIG. 5, the transparent sample cell 2 is held by a positioning mechanism 3 that can be displaced in the XYZ directions, and an objective lens 4 is disposed below it. Light that excites a marker labeled with a fluorescent dye is irradiated from the tip of the optical fiber probe 1, and fluorescence from the sample is collected through the objective lens 4 and then detected by the detector 5. By scanning the positioning mechanism 3 two-dimensionally in the XY direction, the shape of the sample surface and a fluorescent image can be acquired. The detected fluorescent label signal can be subjected to comparative analysis of the position of the fluorescent label on the chromosome and the shape data of the chromosome by the controller 6 which also serves as a control device and a signal analysis device. On the other hand, the shape image of the entire chromosome can be obtained with higher accuracy using the AFM probe 7 than with the optical fiber probe 1, and precise analysis can be performed by the controller 6.
Usually, the shape of the chromosome can be accurately obtained by the AFM probe 7 which also serves as a scraping mechanism, and minute fragments at precise positions on the chromosome can be scraped continuously. In some cases, a specific position on the chromosome is fluorescently labeled by the FISH method, the fluorescence data is acquired by the fiber optic probe 1 to identify the exact scraping position, and the AFM probe 7 is used to scrape accurately. It is also possible to perform. The scraped chromosome fragments are put into the DNA extraction means 8, and the extracted DNA is transferred to the amplification means 9. The amplified DNA is cloned into an appropriate vector, and the base sequence of the clone is sequentially determined by the base sequence determination means 10. The obtained base sequence data is sent to the data processing mechanism 11, and the base sequence of the fragment is restored by comparing and analyzing the base sequence of each clone. In the data processing mechanism 11, the base sequence data of the continuously scraped fragments are connected to restore the base sequence of the entire chromosome.

When creating a physical map, after cloning the DNA amplified by the amplification means 9, the base sequence determination means 10 determines the base sequences of some arbitrarily selected clones, and the primer is designed based on that sequence. Then, PCR amplification is performed by the amplification means 9 using a BAC clone contained in the genomic library as a template, and a BAC clone containing the sequence of the fragment is screened. Based on the information of the screened BAC clone and the position information of the fragment on the chromosome, the data processing mechanism 11 positions the BAC clone on the chromosome and creates a physical map.
The optical fiber probe 1 shown in FIG. 5 is an AFM optical fiber probe bent in the middle, but a shear force system using a straight type optical fiber probe can also be applied. Moreover, although the example which detects the fluorescence of the marker excited with the probe made from an optical fiber through the transparent sample cell 2 was shown, the reflection mode which detects the fluorescence excited from the sample with the probe made from an optical fiber itself, or optical fiber made An illumination / collection mode in which light is collected by an objective lens installed on the probe side is also applicable.
In FIG. 5, the scraping mechanism also serves as the AFM probe 7, but the optical fiber probe 1 can also serve as the scraping mechanism. A separate scraping mechanism can also be provided separately. By providing an XY positioning mechanism with a large moving distance and moving the sample surface, it is possible to scan a large range of several hundred μm square or more, and it is possible to simultaneously analyze many samples on the same sample substrate It is.
Since the detector 5 detects a large number of fluorescent dyes at the same time, it can be combined with a filter set for sequentially transmitting or blocking a specific wavelength or a highly sensitive spectroscopic device.
The controller 6 controls the optical fiber probe 1, positioning mechanism 3, detector 5, AFM probe 7, DNA extraction / amplification means, and records the scraped location information. Alternatively, it can be performed by a recording device.

  According to the present invention, it is possible to perform base sequence decoding and physical map creation in a dramatically shorter period of time and with less labor than conventional methods. As a result, it can be expected to promote genome isolation and variety improvement by genome analysis of various useful animals and plants and its application.

It is a flowchart which shows the operation procedure of the base sequence decoding by this invention. An AFM image (left) showing a state where a region of 100 nm in width of a chromosome was cut and collected, and an example (right) in which DNA derived from fragments was amplified by PCR. It is a flowchart which shows the operation procedure of physical map creation by this invention. It is the schematic diagram which showed the outline of physical map preparation by this invention. It is the schematic diagram which showed the base-sequence decoding and physical map creation apparatus by this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Optical fiber probe 2 Sample cell 3 Positioning mechanism 4 Objective lens 5 Detector 6 Controller 7 AFM probe 8 DNA extraction means 9 DNA amplification means 10 Base sequence determination means 11 Data processing mechanism

Claims (11)

A method for decoding a eukaryotic genome sequence,
Sequentially cleaving eukaryotic chromosomes into multiple microfragments,
Recording the positions of the plurality of microfragments on the chromosome; and collecting the plurality of microfragments individually;
Only including,
further,
A process of extracting DNA from the recovered microfragments,
A step of performing PCR using the extracted DNA as a template and using a primer having a random sequence in part or all thereof,
A step of recovering and cloning the obtained plurality of PCR products to obtain base sequence data of the full length or a part of the plurality of PCR products;
Reconstructing the base sequence of the entire microfragment by joining the base sequence data based on the base sequence of each end of the plurality of PCR products; and
A step of restoring the full length or a part of the base sequence of the chromosome by aligning the base sequence of the restored microfragment based on the record of the position of the plurality of microfragments on the chromosome.
A method for decoding a genomic base sequence, comprising:
The step of cutting the chromosome is performed by scraping with a probe of an atomic force microscope or a near-field optical probe microscope, scraping with a glass or metal needle or capillary, or breaking with a laser,
The step of recording the positions of the plurality of microfragments is performed by using an atomic force microscope, a near-field optical probe microscope, an optical microscope, a transmission electron microscope, or a scanning electron microscope.
The genome base sequence decoding method.   The genomic nucleotide sequence decoding method according to claim 1, wherein the chromosome is a mitotic chromosome or a pachytene chromosome.   The genome base sequence decoding method according to claim 1, wherein the plurality of microfragments have a length of 0.5 µm or less with respect to the chromosome major axis direction.   After scraping the scrape linearly to the center in the direction perpendicular to the long axis of the chromosome, the position is shifted in the major axis direction by 1/20 to 1/2 of the scraping width and remains linearly again. The genome base sequence decoding method according to claim 1, wherein the method is carried out by scraping half of the sample or by scratching at an angle of 5 ° to 30 ° from the direction perpendicular to the major axis. A method of creating a genome physical map of a eukaryote,
Sequentially cleaving eukaryotic chromosomes into multiple microfragments,
Recording the positions of the plurality of microfragments on the chromosome; and collecting the plurality of microfragments individually;
Only including,
further,
A process of extracting DNA from the recovered microfragments,
A step of performing PCR using the extracted DNA as a template and using a primer having a random sequence in part or all thereof,
Recovering and cloning the obtained PCR product and determining the base sequence of the PCR product;
Fragmenting genomic DNA extracted from the eukaryote and inserting it into an arbitrary cloning vector to create a genomic DNA library;
The step of collating the base sequence of the PCR product with the base sequence of a clone contained in the genomic DNA library and determining the position on the chromosome of the clone contained in the genomic DNA library
A genome physical map creation method including:
The step of cutting the chromosome is performed by scraping with a probe of an atomic force microscope or a near-field optical probe microscope, scraping with a glass or metal needle or capillary, or breaking with a laser,
The step of recording the positions of the plurality of microfragments is performed by using an atomic force microscope, a near-field optical probe microscope, an optical microscope, a transmission electron microscope, or a scanning electron microscope.
The genome physical map creation method.   The genome physical map creation method according to claim 5, wherein the chromosome is a mitotic chromosome or a pachytene chromosome.   The genome physical map creation method according to claim 5, wherein the plurality of microfragments have a length of 0.5 μm or less with respect to the chromosome major axis direction.   The method for preparing a genome physical map according to claim 5, wherein the cloning vector is YAC, BAC, or PAC.   The method for preparing a genome physical map according to claim 5, wherein the step of collating the base sequences is performed by PCR or hybridization.   6. The step of collating the base sequence is performed on a part of clones included in the genomic DNA library, or by a PCR product using a clone included in the genomic DNA library as a template. Genome physical mapping method.   In the step of determining the position, in place of clones contained in the genomic DNA library, amplification is performed by PCR from a known gene derived from the eukaryote and a part thereof or cDNA and EST or the genomic DNA of the eukaryote. 6. The method for preparing a genome physical map according to claim 5, wherein any arbitrary DNA or any synthetic DNA having a random sequence is used.