JP4445611B2 - Equipment for polymorphism analysis of repetitive sequences - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus and method for analyzing a polymorphism of a repetitive sequence in a target nucleic acid. The present invention is useful for confirming the presence or absence of a polymorphism in a repetitive sequence.
[0002]
[Prior art]
Due to advances in molecular biology and genetics, a number of genes responsible for hereditary neurological diseases have been identified (1) (numbers in parentheses are for better understanding of the current situation in the field to which the present invention belongs. , Indicates the number of references cited herein, a list of these references is given before the brief description of the drawings). Among them, diseases caused by abnormal extension of triplet repeats (triple repeats), not gene mutations such as base substitutions, deletions, insertions, duplications, etc. (triplet repeat diseases) (2,3) Is newly found. In these diseases, clinical diversity with different symptoms even within the same family, expression promotion phenomenon that becomes younger with each generation, phenomena such as the severity of the next generation depending on the sex of the parent who transmitted the disease are observed And is of interest from a clinical genetic point of view.
[0003]
Based on the region on the gene where the triplet repeat exists, the triplet repeat disease found to date is arranged, and the extension of the triplet repeat is as follows: (1) Fragile X syndrome existing in the untranslated region at the 5 ′ end, Fragile XE mental retardation, (2) bulbar spinal muscular atrophy present in the translation domain, Huntington's disease, spinocerebellar ataxia type 1, dentate nucleus red nucleus, pallidal Louis atrophy, Machado-Joseph disease, There are spinocerebellar ataxia type 2, spinocerebellar ataxia type 6, {circle over (3)} myotonic dystrophy present in the untranslated region at the 3 ′ end, and {circle over (4)} freetrich ataxia present in the intron.
[0004]
The number of repeats in the causative genes of these diseases is distributed in a certain range of about 40 repeats or less in healthy individuals, and often exhibits a remarkable polymorphism within this range. On the other hand, the increase in the number of repeats in patients is about 40 to 100 repeats in the case of triplet repeats existing in the translation region of the protein, whereas in the case of triplet repeats existing in the untranslated region or intron Shows a marked increase of about 200 to 1000 repeats or more. Regarding the physiological functions of these causative genes, bulbar spinal muscular atrophy is an androgen receptor, and spinocerebellar ataxia type 6 is voltage-dependent Ca.²⁺Channel alpha_1AUnknown except that it is known to encode a subunit.
[0005]
There are the following methods for measuring the number of repetitions of repetitive sequences of genes causing these diseases.
[0006]
First, the most classic method is a method using Southern blot, and the outline is as follows.
[0007]
(1) Cleave genomic DNA sections with known restriction enzymes to produce many DNA fragments.
[0008]
(2) After the DNA fragment is fractionated by gel electrophoresis, the obtained electrophoresis pattern is Southern blotted on a nylon membrane (4).
[0009]
(3) A labeled DNA probe capable of specifically hybridizing with the target gene is hybridized with the Southern blotted DNA fragment.
[0010]
(4) By detecting the band on the electrophoresis pattern to which the labeled probe is bound
Thus, the number of repetitions of the repetitive sequence of the target gene is determined (5).
[0011]
In addition, as one aspect of polymorphism analysis by PCR, methods for determining the number of repetitions of repetitive sequences are also actively performed. In this method, after amplifying the sequence using a primer capable of amplifying the repetitive sequence to be detected, the amplified product is electrophoresed, and the product is detected using staining or autoradiography (6) .
[0012]
However, since both analytical methods require complicated experimental operations like the electrophoresis method and the blotting method, much labor and time are required. In addition, since both blotting and PCR methods use electrophoresis, it is difficult to process multiple samples and multiple items, and it is possible to detect a single base difference in order to accurately measure the number of repetitions. Electrophoresis with extremely high accuracy must be performed. Furthermore, as a label for detecting the gene of interest,³²In the case of using a radioactive isotope such as P, there is also a problem that the number of operators is limited by related laws and regulations.
[0013]
On the other hand, as a completely different method from these analytical methods, in recent years, a method of synthesizing oligonucleotides on a substrate called a gene chip (microarray) (7), a method of spotting PCR products or plasmid DNA on a slide glass ( 8) was developed. In these methods, about 10,000 clones are arrayed, and application to gene analysis such as determination of gene nucleotide sequence, single nucleotide polymorphism, gene hunting, gene expression monitoring and the like has been attempted. These methods have the advantage that a large number of specimens can be detected at the same time because they can be measured efficiently with many types of DNA probes.
[0014]
However, unlike the case of detecting a specific nucleic acid from a group of nucleic acids having a large difference in base sequence, the polymorphism of each repetitive sequence is different only in the number of repeats. A complicated means for finely adjusting the association / dissociation state between each repetitive sequence and the probe is necessary, but such precise control is not possible with the above-described method. Further, since the measurement is performed in a dry state, there is a disadvantage that a change in hybridization cannot be detected in real time.
[0015]
[Problems to be solved by the invention]
This invention is made | formed in view of the said situation, and it aims at providing the apparatus and method which can perform the polymorphism analysis of many repetitive sequences easily.
[0016]
[Means for Solving the Problems]
In order to solve the above problems, the present invention provides:
An apparatus for analyzing a polymorphism of a repetitive sequence,
A substrate provided with a reaction part capable of contacting a liquid sample containing a target nucleic acid having a predetermined repetitive sequence;
A nucleic acid probe comprising a nucleic acid immobilized on the surface of a predetermined region of the reaction part and having a sequence complementary to the predetermined repetitive sequence;
A dissociation processing means for performing a dissociation process on the liquid sample in order to dissociate the target nucleic acid from the nucleic acid probe after hybridizing the nucleic acid probe under a condition capable of hybridizing the target nucleic acid; And a control means for changing the dissociation conditions by the dissociation processing means,
Provided is an apparatus for confirming the presence or absence of a polymorphism of the repetitive sequence based on a difference in dissociation conditions according to the number of repetitive units in the target nucleic acid.
[0017]
Here, the “repetitive sequence” means a nucleic acid sequence in which the same or very similar “repeating unit” (AGT in the right example) is present repeatedly as in AGTAGTAGT. The repetitive sequence includes a tandem repetitive sequence in which a spacer region is interposed between repetitive units and a tandem repetitive sequence in which the repetitive units are directly adjacent to each other without interposing a spacer region between the repetitive units. In the present specification, the “repetitive sequence” includes both.
[0018]
In addition, “polymorphism” means a genetic polymorphism, that is, a plurality of alleles at one locus, and “analyze polymorphism” and “polymorphism analysis” It means performing an analysis to determine the genotype of an allele present at a locus, and that analysis. Therefore, “polymorphism analysis” in a repetitive sequence means analysis to determine the number of repeats of a repeat unit.
[0019]
“Hybridization” means complementary binding that occurs between complementary sequences of so-called nucleic acids (DNA, RNA, etc.). Further, “dissociation” means that a target nucleic acid and a nucleic acid probe which have been once hybridized are separated from each other by releasing complementary binding related to hybridization. Further, the “dissociation treatment” means a treatment necessary for dissociation, for example, heating, alkalinization treatment or the like. In addition, “changing the dissociation conditions” means changing the strength of the dissociation treatment so that the possibility of dissociation varies depending on the number of repetitive sequences in the target nucleic acid. The strength of the dissociation treatment includes a method of changing the concentration of a substance necessary for dissociation or changing the temperature. The dissociation conditions can be changed by reducing the intensity of the dissociation treatment and comparing them, but conversely, increasing the intensity is preferable because the result of changing the conditions can be easily measured.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the present invention will be described below.
[0021]
[Example 1]
In this example, an apparatus for analyzing a polymorphism of a repetitive sequence using a high-density DNA capillary array module capable of delicately controlling the temperature of a liquid sample will be described.
[0022]
As shown in FIG. 1 and FIG. 2, the apparatus of this embodiment is arranged on a substrate 1, a narrow groove 2 for flowing a liquid sample provided on the surface of the substrate 1, and one end of each narrow groove 2. The sample inlet 3 provided, the sample outlet 4 connected to the joint channel formed by joining the ends of the narrow grooves located on the side opposite to the end where the sample inlet 3 is disposed, and the narrow groove 2 A nucleic acid probe 5 solid-phased in a predetermined region, a thin film 6 placed on the bottom of the substrate 1, a heating means 7 and a temperature control means 8 embedded in the thin film.
[0023]
The substrate 1 can be made of a material suitable for microfabrication, preferably glass, silicon single crystal plate, amorphous such as quartz and pyrex, metal such as aluminum and stainless steel, plastic such as fluororesin and polypropylene. A silicon single crystal plate is most preferable because it can be easily processed by a known semiconductor process technology.
[0024]
The substrate 1 is preferably rectangular from the viewpoint of ease of processing, but may be any shape such as polygons such as rhombus, hexagon, and octagon, circle, and ellipse.
[0025]
The substrate 1 can have any size, but when the shape of the substrate 1 is a rectangle, the long side is preferably 0.1 to 15 cm and the short side is preferably 0.1 to 15 cm.
[0026]
The thickness of the substrate 1 can be 0.01-0.5 cm.
[0027]
The narrow groove 2 is provided on the surface of the substrate 1, and a liquid sample containing a predetermined repetitive arrangement is poured into the narrow groove 2.
[0028]
When the substrate is a silicon single crystal plate, the narrow groove 2 can be formed by a well-known etching process in the semiconductor manufacturing process. When glass is used as the substrate, the narrow groove 2 can be formed by etching using hydrofluoric acid. Even when other materials are used as the substrate, the narrow groove 2 can be formed using a known technique such as etching. It is preferable to join one end of each narrow groove to form a combined flow path so that the liquid sample poured into the narrow groove 2 can be efficiently recovered.
[0029]
The dimensions of the narrow groove 2 may be any dimensions depending on the volume and fluidity of the liquid sample to be measured, but are preferably 5 to 100 mm in length, 1 μm to 10 mm in width, and 0.1 to 50 mm in depth.
[0030]
It is preferable to form 10 to 10,000 narrow grooves 2 at intervals of 0.01 to 50 mm so that a large amount of specimen can be measured simultaneously.
[0031]
The sample inlet 3 is provided as an opening for injecting the liquid sample into the narrow groove 2 by manual or automatic appropriate dispensing means, and the sample outlet 4 is provided by a desired suction means. It is provided as an opening for discharging each liquid sample poured into the narrow groove 2.
[0032]
Since the narrow groove 2 can be very thin, if the sample inlet 3 and the sample outlet 4 are provided, the liquid sample can be easily injected and discharged.
[0033]
The sample inlet 3 and the sample outlet 4 are preferably made of materials that are difficult to nonspecifically adsorb substances in the sample such as nucleic acids. Teflon tubes and polypropylene tubes are suitable materials because of less non-specific adsorption.
[0034]
The nucleic acid probe 5 is a group of nucleic acids containing a nucleic acid having a sequence complementary to a predetermined repetitive sequence contained in a liquid sample. In the present specification, “nucleic acid” includes both DNA and RNA.
[0035]
A method for immobilizing a nucleic acid probe in the narrow groove 2 is disclosed in, for example, Science 251: 767-773 (1991). The outline of the method is as follows: (1) A silicon substrate is subjected to silane treatment to form an amino group on the substrate, and a photoprotective molecule is bonded to each amino group.
(2) Use a mask to selectively irradiate the desired position with ultraviolet rays, remove the photoprotective molecule at the desired position, and expose only the amino group at the position. (3) Exposed amino group Only the desired nucleobase linked to the photoprotective molecule is bound, and the steps (4), (2) and (3) are repeated to extend the nucleic acid probe.
[0036]
Also, other improved methods for immobilizing nucleic acid probes are disclosed in International Publication No. WO93 / 09668 (Japanese Patent Publication No. 7-506561) and International Publication No. WO99 / 11754.
[0037]
The nucleic acid probe is immobilized on a predetermined region of the narrow groove at an appropriate density according to the volume of the liquid sample to be injected and the nucleic acid content of the liquid sample. As shown in FIG. 1, the nucleic acid probe may be immobilized on a plurality of locations in the narrow groove at intervals of about 1 μm to 50 mm as necessary.
[0038]
If the repeat unit of the predetermined repetitive sequence to be subjected to polymorphism analysis is 3 bases, the nucleic acid probe 5 may be a group consisting of 20 types of combinations described in FIG.
[0039]
For example, TTG / TTG / TTG / TTG / TTG repeat units are TTG, TGT, GTT Note that the repeat unit is identical to the repeat unit TT / GTT / GTT / GTT / GTT / G as the repeat unit), and the sequence complementary to the repeat unit is AAC, ACA, CAA. Since it is a repetitive sequence, the repeat unit of 3 bases is a combination of 4 bases 4^Three= 64, there are 20 types obtained by dividing 60 combinations obtained by subtracting 4 combinations of AAA, TTT, CCC, and GGG and dividing by 3. Therefore, if the repeat unit of a predetermined repetitive sequence to be subjected to polymorphism analysis is 3 bases, it is possible to analyze polymorphism of any repetitive sequence by immobilizing up to 20 kinds of nucleic acid probes. It becomes possible. According to the same calculation, if a nucleic acid probe of 60 types for a repeating sequence having 4 bases and 204 types for a repeating sequence of 5 bases is immobilized on the narrow groove 2, any repetitive sequence can be obtained. It is clear that polymorphisms can be analyzed. That is, if the sequence to be subjected to polymorphism analysis is a repetitive sequence, there is a great advantage that the number of nucleic acid probes to be immobilized is significantly less than the analysis of a general sequence consisting of a disordered base sequence combination. .
[0040]
As described above, in an apparatus for performing polymorphism analysis of a repetitive sequence having 3 bases as a repeating unit, 20 types of nucleic acids may be immobilized on one narrow groove, but as shown in FIG. It is also possible to provide a plurality of regions where the nucleic acid is immobilized. Nucleic acid probes with different numbers of bases of repeating units may be immobilized on each narrow groove of one apparatus. Alternatively, nucleic acid probes having different numbers of bases of repeating units may be immobilized on different regions of one fine groove.
[0041]
The number of repeating units in the nucleic acid probe, that is, the number of repetitions may be any number of 2 or more, but in view of the fact that the number of repetitions of the repetitive sequence present in the living body is usually 2 to 6, appropriate analysis In order to implement, it is preferable that it is 2-6.
[0042]
The thin film 6 is disposed at the bottom of the substrate 1 and has heating means 7 and temperature control means 8 embedded therein. The material of the thin layer 6 can be any material having excellent heat resistance and high strength. The apparatus of the present invention preferably has heat resistance up to about 100 ° C. Materials suitable for use in the thin layer 6 are polyimide, polypropylene resin, vinyl chloride resin, fluororesin and the like, and polyimide is particularly preferable from the viewpoint of heat resistance and strength.
[0043]
The heating means 7 has a function of heating the liquid sample injected into the narrow groove. Therefore, the heating means needs to be embedded in the thin film so that the liquid sample in the narrow groove can be heated. The heating means may be any member that can achieve the function of heating the liquid sample. As an example, there may be mentioned one having a metal such as nickel chrome or titanium and a voltage load means for energizing the metal. It is preferable to arrange a plurality of heating means at equal intervals in the thin film so that the liquid sample in the narrow groove is uniformly heated.
[0044]
The temperature control unit 8 includes a temperature detection unit and a temperature control unit, detects the temperature of the liquid sample heated by the heating unit 7, and prevents further heating when the temperature reaches a desired temperature. Has a function of stopping heating by the heating means 7. Therefore, the temperature control means needs to be embedded in the thin film so that the liquid temperature in the narrow groove can be detected and the liquid temperature can be controlled.
[0045]
The temperature control means is a micro for controlling the voltage to the heating means so that the temperature of the liquid sample becomes a desired temperature by converting the resistance value into a temperature and a metal whose resistance value linearly changes with temperature. It can be a general one with a computer.
[0046]
It is preferable to arrange a plurality of temperature control means at equal intervals so that the liquid sample in the narrow groove is kept at a uniform temperature.
[0047]
This example also discloses a method for analyzing a polymorphism of a predetermined repetitive sequence contained in a liquid sample. Specifically, when the above-described apparatus is used, the following operation can be performed.
[0048]
(1) A liquid sample containing a target nucleic acid having a predetermined repetitive sequence is prepared.
[0049]
(2) The target nucleic acid is labeled as necessary.
[0050]
(3) A liquid sample is injected from the sample injection port 3 and the labeled target nucleic acid is hybridized to the nucleic acid probe. In this apparatus, complementary binding to the nucleic acid probe 3 occurs at a stage where the double-stranded structure of the target nucleic acid is dissociated into a single-stranded figure after returning to a double-stranded structure by temperature control. Immediately after the hybridization, preferably, in order to remove the non-hybridized target nucleic acid, a washing solution (eg, physiological saline, buffer solution, ion-exchanged water, etc.) having a composition that does not affect hybridization is further added. The sample is injected and discharged from the sample discharge port 5. However, it is preferable that the cleaning liquid is preheated to a temperature equivalent to the temperature condition by the temperature control means 9 so as not to prolong the temperature control.
[0051]
(4) Heat the liquid sample to a desired temperature. Here, preferably, in order to remove the target nucleic acid that has dissociated upon reaching a temperature corresponding to the dissociation condition according to the number of repetitive sequences, the above-described washing solution is further injected and discharged from the sample discharge port 5. When multiple dissociation conditions are set, the above-mentioned washing may be removed at each different heating temperature, or the temperature is continuously increased while continuously injecting the above-mentioned washing solution to improve efficiency. You may plan.
[0052]
(5) Analyzing polymorphisms of the repetitive sequence based on the difference in dissociation temperature according to the number of repetitive units of the repetitive sequence
A “liquid sample” is a biological material that is itself liquid, or a biological material dissolved or suspended in a suitable liquid, containing the desired repetitive sequence to be subjected to polymorphic analysis. It is a sample considered to be. The liquid sample may be one obtained by dissolving or suspending body fluid such as blood, saliva or semen, blood cell components, biopsy tissue, cultured cells, etc., but is not limited thereto.
[0053]
A “target nucleic acid” can be any nucleic acid known to have a repetitive sequence, but in particular, a polymorphism of the repetitive sequence can be a marker for the presence or absence of disease or a disease probability, or a marker for genetic analysis. It is preferred that the nucleic acid is clear. Many repetitive sequences exhibit significant polymorphism, and are highly useful as disease markers and genetic analysis markers. Therefore, there are many nucleic acids having such repetitive sequences. If the repetitive sequence is a disease marker, data for diagnosing a disease whose polymorphism of the repetitive sequence can be an indicator of the presence or absence of a disease or a disease probability can be acquired.
[0054]
Each causative gene of triplet repeat disease as described above is a particularly preferred target nucleic acid. Microsatellite DNA is particularly useful for use in genetic analysis and individual identification. Each causative gene of triplet repeat disease has 3 bases, and microsatellite DNA has 1 to 5 bases.
[0055]
The target nucleic acid is labeled with a fluorescent label, a radioactive label or the like as necessary.
[0056]
A liquid sample containing the target nucleic acid is injected into the narrow groove from the sample injection port, and the target nucleic acid is hybridized with a nucleic acid probe having a sequence complementary to a repetitive sequence in the nucleic acid.
[0057]
After hybridizing the target nucleic acid to the nucleic acid probe, the liquid sample is heated to the desired temperature. Since a nucleic acid having a larger number of repeating units in a repetitive sequence binds more strongly to a nucleic acid probe, a nucleic acid having a larger number of repeating units is less likely to dissociate than a nucleic acid having a smaller number of repeating units. Therefore, by using such a difference in dissociation temperature, it is possible to analyze a polymorphism of a repetitive sequence in a target nucleic acid, and to diagnose a disease that can be used as an indicator of the onset presence probability or the probability of occurrence. You can get data to do. That is, “analyzing a polymorphism of a repetitive sequence” “based on the difference in dissociation temperature” means that polymorphism analysis is performed by an operation using a difference in dissociation temperature of repetitive sequences having different numbers of repeating units. Specifically, operations as shown in FIGS. 4 to 6 can be considered, but the present invention is not limited to these operations.
[0058]
Hereinafter, an operation for measuring the number of repeating units using the difference in dissociation temperature will be described with reference to FIGS.
[0059]
  Figure 4 shows the most basic operation, labeled abnormal target nucleic acid.11And normal target nucleic acid12(In the figure, the label is indicated as F)13After binding to normal target nucleic acid12Only specifically dissociate. Thus normal target nucleic acid12Dissociation of only the target level decreases the background level, so the sensitivity of the measurement increases significantly.11It is possible to easily measure whether or not is included.
[0060]
In a 1 mM Na (0.006 × SSC) solution, the dissociation temperature is determined by the following equation.
[0061]
Dissociation temperature = 81.5 ° C. + 16.6 logs (Na concentration (M)) + 0.41 × (% GC content) −675 / sequence length−0.65 × (% formamide) − (% mismatch)
In the operation of FIG. 5, a hybrid of an abnormal target nucleic acid 1 and a normal target nucleic acid 2 and a labeled primer 3 is bound to the nucleic acid probe 4, and then heated to form a normal target nucleic acid 2 and a labeled primer 3. Only the hybrid is specifically dissociated from the nucleic acid probe 4.
[0062]
  As is clear from the figure, the primer23Normal target nucleic acid22If set to be longer, normal target nucleic acid22All or most of the sequence of23Probe that hybridized with and immobilized24Cannot bind to the primer, but even if bound, the primer23Short protrusions that are not paired with only form a weak bond. In contrast, the abnormal target nucleic acid 1 is a primer.23Longer than the primer23Long protrusions that do not mate with the primer23And can be firmly bonded. Therefore, normal nucleic acid22And primer23Hybrids with abnormal nucleic acids21And primer23Dissociates at a lower temperature than the hybrid.
[0063]
  In the operation of FIG. 6, the abnormal target nucleic acid31And normal target nucleic acid32Shorter nucleic acid probes33In addition, abnormal target nucleic acid31Or normal target nucleic acid32After hybridizing a part of the labeled nucleic acid34Add a nucleic acid probe33Target nucleic acid that did not hybridize with31Or normal target nucleic acid32Labeled nucleic acid in the remainder of33Is hybridized. As shown in the figure, abnormal target nucleic acid31Is the normal target nucleic acid32More labeled nucleic acid because it is longer than34Can hybridize. Subsequently, labeled nucleic acid34If the background value is lowered by heating near the temperature at which dissociation occurs, abnormal target nucleic acid31And normal target nucleic acid32Even if the difference in the number of iterations is small, both can be identified and the number of iterations can be determined.
[0064]
As described above, by confirming the presence or absence of a polymorphism of a repetitive sequence in a specimen (blood, tissue extract, etc.) collected from a subject, various pathological conditions, genetic character, etc. resulting from the polymorphism of the repetitive sequence It is possible to obtain data capable of diagnosing a disease or personality that can be used as an index of the presence or absence of the genetic feature of the present invention or the probability of occurrence or onset of the genetic feature. In addition, this invention is not limited to the Example mentioned above, A various obvious change is possible by applying a well-known genetic engineering technique. For example, in the above-described embodiment, a predetermined amount of the nucleic acid probe is immobilized on the surface of the substrate, but it is hybridized without immobilizing it on the substrate, and image analysis or observation evaluation of the movement state or distribution state of the molecule is performed. By doing so, the analysis can be executed. In addition, when the washing step as described above is performed after the hybridization and the dissociation step, the analysis is performed by measurement without using a label (for example, ellipsometer, scanning atomic microscope, etc.). Also good.
[0065]
By using the apparatus and method for repetitive sequence polymorphism analysis according to the present invention, a polymorphism of a target nucleic acid having a repetitive sequence having a different number of repetitions can be easily identified and determined. This apparatus and method uses the fact that the temperature at which the target nucleic acid is dissociated from the hybrid formed with the complementary nucleic acid differs if the number of repeats of the repetitive sequence contained in the target nucleic acid is different. Although the polymorphism is determined, since the present apparatus and method can use a sample in a solution state, the temperature of the sample can be precisely controlled so that a slight difference in the number of repetitions can be detected.
[0066]
In addition, the polymorphism analysis of repetitive sequences using the apparatus and method of the present invention is much easier to operate than conventional methods using electrophoresis, blotting, and PCR, and many samples can be obtained. It has a very remarkable effect that it can be measured at once.
[0067]
【The invention's effect】
  According to the apparatus and method of the present invention, polymorphisms having different numbers of repetitions can be easily analyzed for target nucleic acids having repetitive sequences. Moreover, it is possible to detect a change in hybridization according to the number of repetitions in real time while changing the dissociation conditions, and the operability is excellent.Furthermore, the presence and the number of iterations can be confirmed even if the difference in the number of iterations is small.
[0068]
[References]
(1) Martin, J.B .: Ann. Neurol., 34, 757-773 (1993)
(2) La Spada, A.R .: Ann. Neurol., 36, 814-822 (1994)
(3) Ross, C.A.:Neurol., 36, 814-822 (1994)
(4) Southern E.M .: J. Mol. Biol., 98, 503 (1975)
(5) Verkerk, A.J.M.H.:Cell, 65, 905-914 (1991)
(6) Fu, T.:Cell, 67, 1047-1058 (1991)
(7) Fordor, S.P.A.:Science, 251, 767-773 (1991)
(8) Schena, M .: Science, 270, 460-470 (1995)
[Brief description of the drawings]
FIG. 1 is a perspective view of an apparatus of the present invention.
2 is a longitudinal sectional view of the device of the present invention taken along line l-l ′ in FIG. 1;
FIG. 3 is a view showing combinations of nucleic acid probes to be immobilized on the apparatus of the present invention in order to analyze a polymorphism of a target nucleic acid having a repetitive sequence consisting of a repeating unit of 3 bases.
FIG. 4 is a diagram showing one embodiment of a method for analyzing polymorphisms of repetitive sequences using the apparatus of the present invention.
FIG. 5 is a view showing a further embodiment of a polymorphism analysis method for repetitive sequences using the apparatus of the present invention.
FIG. 6 is a diagram showing a further embodiment of a method for analyzing polymorphisms of repetitive sequences using the apparatus of the present invention.

Claims (3)

A method of analyzing polymorphisms for repeated sequences with an increased number of repeats than normal ,
(1) Solid phase containing a target nucleic acid having a predetermined repetitive sequence to be analyzed, which is a nucleic acid having a sequence complementary to the predetermined repetitive sequence and having a smaller number of repeats than a normal target nucleic acid Hybridizing by mixing under the conditions capable of hybridizing with the activated nucleic acid probe ;
(2) After the hybridization, a labeled nucleic acid that is labeled and binds to the target nucleic acid according to the number of repetitions is added to the single-stranded portion of the target nucleic acid that is not hybridized with the nucleic acid probe. Applying a dissociation treatment to the mixture hybridized with
(3) For the dissociated mixture, repeating the dissociation process with different dissociation conditions; and
(4) A method comprising determining the presence / absence of a polymorphism in the repetitive sequence and the number of repeats based on the difference in the dissociation conditions and a signal from the labeled nucleic acid .   The method according to claim 1, wherein the dissociation condition is a dissociation temperature.   The method according to claim 1 or 2, wherein the change of the dissociation condition is to increase the strength of the dissociation condition.

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