KR20070062480A - Microarray and kit for diagnosing cmt1a - Google Patents

Abstract

A microarray and kit for diagnosis of CMT1A(Charcot-Marie-Tooth neuropathy 1A) syndrome are provided to detect chromosome disorder resulting in CMT1A syndrome, especially chromosome 17 DNA duplication in the total of human genomic DNA rapidly and accurately. The microarray for diagnosis of CMT1A syndrome comprises at least one probe selected from (a) at least one genomic DNA fragment selected from BAC(bacterial artificial chromosome) DNA Nos. 1-235, (b) a 20-100 bp oligonucleotide continuously positioned in the genomic DNA fragment, (c) a 100bp-10kbp cDNA(complementary DNA) derived from the genomic DNA fragment, and (d) a complementary gene of the genomic DNA fragment, oligonucleotide and cDNA, wherein the content ratio of repeated sequence in the nucleotide sequence of the probe is up to 85%; the nucleotide sequence of the probe has a single locus in the chromosome; the probe is produced by inserting the genomic DNA fragment into a vector selected from BAC, HAC(human artificial chromosome), TAC(transformation competent artificial chromosome), YAC(yeast artificial chromosome), PAC(P1-derived artificial chromosome), P1(phage) and cosmid, and is immobilized to the microarray at a concentration of 2-100 pg per spot; and at least two kinds of probes are immobilized per one spot. The kit for diagnosis of CMT1A syndrome comprises the microarray and a marker material-detecting tool, and determines chromosome disorder of a testing sample by reacting the total genomic DNA of testing sample labeled with a first probe detectable marker material with the standard DNA labeled with a second probe detectable marker material, and measuring each hybridization ratio of the testing sample DNA and standard DNA, wherein the first and second marker materials are each independently a radioactive isotope, a fluorescent material, a chemiluminescent or an enzyme.

Description

CMTIA Diagnostic Microarrays and Kits {MICROARRAY AND KIT FOR DIAGNOSING CMT1A}

1 is a cleavage map of the pECBAC1 vector.

Figures 2a to 2h shows the intrachromosomal location of the probe used in the BAC chip of Example 2.

3 shows the configuration of the BAC chip of the second embodiment.

4a to 4f show probe positions specific to each chromosome in the BAC chip of Example 2. FIG.

Figure 5 shows the results of BAC chip analysis of women with Patau syndrome.

Figure 6 shows the results of the BAC chip analysis of male patients with Patau syndrome.

Figure 7 shows the results of the BAC chip analysis of Edward's male diseased.

Figure 8 shows the results of BAC chip analysis of Edward syndrome female disease.

Figure 9 shows the results of BAC chip analysis of women with Down syndrome.

Figure 10 shows the results of BAC chip analysis of a female with chromosome 21 amplified, X X chromosome amplified one more karyotype XXY.

Figure 11 shows the results of BAC chip analysis of Turner syndrome disease.

Figure 12 shows the results of BAC chip analysis of Klinefelter syndrome disease.

Figure 13 shows the results of BAC chip analysis of normal women.

Figure 14 shows the results of BAC chip analysis of a normal male.

Figure 15 shows the results of BAC chip analysis of Down syndrome female disease.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a method for assaying chromosomal abnormalities, in particular, CMT1A (Charcot-Marie-Tooth neuropathy 1A), and more particularly, by quickly and accurately assaying chromosomal abnormalities, it is possible to easily diagnose the state of health and disease of a specimen. The present invention relates to a method for detecting a chromosome abnormality capable of identifying a disease-specific chromosome abnormality and providing an indicator for diagnosing a disease.

[Private Technology]

Chromosomal aberrations are associated with genetic disorders and degenerative diseases and represent defects or duplications of individual chromosomes, loss or duplication of chromosomal portions, chromosomal breaks, inversions and translocations, and the like. Chromosomal aberrations can disrupt the genetic balance of an organism, causing fetal death or severe mental and physical coupling. Down syndrome, for example, is caused by the presence of chromosome 21 in multiples of 3 and is a representative disorder of chromosomal abnormalities. In addition, Edwards syndrome (18+), Patau syndrome (13+), Turner syndrome (XO) and Klinefelter syndrome (XXY) are among the chromosomal abnormalities.

Chromosomal aberrations are diagnosed by karyotype and Fluorescent In Situ Hybridization (FISH). The methods are time consuming and labor intensive compared to accuracy, and particularly karyotyping requires a cell culture time. In addition, FISH has a disadvantage in that the analyte is limited only to the conventional sequencing and chromosome positional properties. As a solution to the above-mentioned disadvantages of FISH, there is a comparative genome hybridization (CGH). CGH analyzes the entire genome to detect sites that show more than the number of chromosomes. However, CGH has a disadvantage of lower resolution than FISH.

Alternatively, a DNA microarray system can be used. DNA microarray systems are classified into cDNA microarrays, oligonucleotide microarrays and genomic microarrays according to bio-molecules integrated on the array. cDNA microarrays and oligonucleotide mitroarrays have the advantage of being easy to fabricate, but have the disadvantage of limiting the number of probes that can be immobilized on the array, the cost of making excessive probes, and chromosomal abnormalities in other parts of the probe. In particular, genomic DNA microarrays, on the other hand, facilitate the preparation of probes, can detect chromosomal abnormalities in a wide range of chromosomes, and can detect abnormalities in intron sites within chromosomes. There is a disadvantage that it is difficult to prepare a large number of DNA fragments which are located.

On the other hand, BAC (Bacterial Artificial Chromosome) clone is a strain containing a BAC vector in which the entire genome of a human is inserted in a certain size, and each strain contains one human DNA fragment. And by analyzing the location on the chromosome, one can enumerate the BAC clones corresponding to the DNA fragments of the specific chromosome site. Therefore, there is an advantage that a specific DNA fragment can be easily obtained from each BAC clone.

In order to solve the problems of the prior art, an object of the present invention is to provide a method for producing genomic DNA microarray for chromosomal abnormality assay.

It is another object of the present invention to provide a microarray manufacturing method in which two or more probes are fixed to one spot.

It is another object of the present invention to provide a DNA chip for chromosomal aberration assay for the entire human genome.

In addition, the present invention, Down syndrome, Patau syndrome, Edward syndrome, Turner syndrome, Kleinfelter syndrome, ATR-16 (alpha-thalassemia retardation) syndrome, CAT1A (Charcot-Marie-Tooth neuropathy 1A), Cat cry syndrome, De George Syndrome, Hereditary Neuropathy with liability to presure palsies disease, Kalman syndrome, Miller-Dicker syndrome, Prader-Willi syndrome, Rubinsteintabi syndrome, Smith-Magenes syndrome, steroid sulfatase gene syndrome, Williams syndrome, Wolf An object of the present invention is to provide a DNA chip for assay for a disease selected from the group consisting of Hirschhorn syndrome and prenatal-postnatal chromosomal abnormalities.

Another object of the present invention is to provide a method for detecting chromosomal abnormalities using a microarray for detecting chromosomal abnormalities.

Another object of the present invention is to provide a method for diagnosing chromosomal disorders using a microarray for detecting chromosomal abnormalities.

Another object of the present invention is to provide a method for detecting a chromosomal abnormality specific to a disease using a microarray for detecting a chromosomal abnormality.

The present invention to achieve the above object

(a) identifying the genomic DNA fragments inserted into each clone from the clones obtained from the genomic library of the organism,

(b) selecting a target chromosome region to check for chromosomal abnormalities;

(c) selecting genomic DNA fragments capable of detecting the target chromosomal region as probes among the genomic DNA fragments identified in step (a),

The probe has a maximum content of 85% repeats in the nucleotide sequence and the nucleotide sequence constituting the probe includes a single locus in the chromosome, and the probe includes all, part or the complement thereof of the target chromosome region. A sequence, and

(D) provides a method for producing genomic DNA microarray for detecting chromosomal abnormalities comprising the step of fixing the probe to a microarray.

The present invention also provides a microarray manufacturing method comprising the step of immobilizing a probe selected from the group consisting of RNA fragments, cDNA fragments, oligomers and genomic DNA fragments in a microarray, wherein the microarray is two or more per one spot Wherein the two or more probes immobilized in the one spot comprise a sequence selected from the group consisting of all, a portion of the chromosomal region and their complements that exhibit the same gene phenotype in a chromosomal abnormality disease Phenotypes provide microarray preparation methods that are gene amplification or gene deletion.

The present invention also provides a method for immobilizing one or more genomic DNA fragments selected from human genomic genes, one or more oligomers of 20 to 100 bp contiguous in the fragments, or one or more cDNAs of 100 bp to 10 kbp derived from the fragments. A DNA chip for chromosomal aberration assay for the entire human genome including a microarray is provided.

In addition, the present invention, Down syndrome, Patau syndrome, Edward syndrome, Turner syndrome, Kleinfelter syndrome, ATR-16 (alpha-thalassemia retardation) syndrome, CAT1A (Charcot-Marie-Tooth neuropathy 1A), Cat cry syndrome, De George Syndrome, Hereditary Neuropathy with liability to presure palsies disease, Kalman syndrome, Miller-Dicker syndrome, Prader-Willi syndrome, Rubinsteintabi syndrome, Smith-Margenes syndrome, steroid sulfatase gene syndrome, Williams syndrome, Wolf A DNA chip for assay for a disease selected from the group consisting of Hirschhorn syndrome and prenatal-postnatal chromosomal abnormalities is provided.

In another aspect, the present invention (a) preparing a microarray for detecting chromosomal abnormalities, (b) mixed reaction of the labeled sample genomic DNA and the labeled standard DNA in the same ratio to the probe immobilized on the microarray, It provides a method for detecting chromosomal abnormalities comprising the step of measuring the hybridization ratio of each of the sample DNA and the standard DNA on the microarray, and (c) determining the chromosomal abnormality of the sample DNA from the measurement result of the hybridization ratio.

In another aspect, the present invention (a) selecting a probe comprising a continuous 20 to 100 bp in genomic DNA fragments and fixing it on a microarray,

The genomic DNA fragment is selected from the DNAs listed in Table 1 below.

The probe has a maximum content of 85% repeat sequence in the nucleotide sequence

The base sequence constituting the probe includes a single locus in the chromosome

(b) the DNA layer on the microarray is mixed with the sample genomic DNA labeled with the detectable first label and the standard DNA labeled with the detectable second label at the same ratio, and the sample DNA and the standard on the microarray. Measuring the hybridization rate of DNA and

(c) From the measurement result of the hybridization ratio, when the hybridization ratio of the sample DNA is the same as the hybridization ratio of the standard DNA, it is read that there is no chromosomal abnormality, and when the former is higher than the latter, the chromosomal increase is read, and the former It provides a method for detecting chromosomal abnormalities comprising the step of analyzing the presence of chromosomal defects when compared to the latter.

In another aspect, the present invention is (a) fixing a probe selected from the group consisting of RNA fragments, cDNA fragments, oligomers and genomic DNA fragments in a microarray,

The microarray includes two or more probes per spot,

Probes having the same sequence of different sequences immobilized in the one spot include sequences selected from the group consisting of all, a portion of the chromosomal sites and their complements which exhibit the same gene phenotype in a disease of chromosome number or more;

The gene phenotype is amplification or deletion

(b) mixing and reacting the sample sample labeled with the detectable first labeling substance and the standard sample labeled with the detectable second labeling substance in the same ratio to the prepared microarray, and hybridizing the sample sample and the standard sample on the microarray. Measuring the ratio and

(c) As a result of the measurement, if the hybridization ratio of the sample sample and the standard sample is the same, no chromosomal abnormality is read; if the former is higher than the latter, the chromosome amplification is read; if the former is lower than the latter, the chromosome The present invention provides a method for diagnosing a disorder of chromosomal abnormalities, including analyzing a defect and determining a chromosomal abnormality of a specimen sample.

In addition, the present invention (a) identifying each genomic DNA fragment inserted into the clone from each of the clones obtained from the genomic library of the organism, (b) all or part of the genomic DNA fragments identified in step (a) Immobilizing on the microarray, (c) mixing the sample genomic DNA labeled with the detectable first label of the diseased patient and the standard DNA labeled with the detectable J. label with the same ratio to the probe immobilized on the microarray. Reacting, determining the hybridization rate of the sample DNA and the standard DNA, and (d) analyzing the hybridization rate to identify the site and the chromosomal abnormality form indicating the chromosomal abnormality of the diseased patient. Provided are methods for detecting chromosomal abnormalities.

Hereinafter, the present invention will be described in detail.

As used herein, the term "organism" refers to a plant, insect, animal or human.

The "genomic DNA microarray" referred to in the present invention is a microarray in which genomic DNA fragments of an organism are fixed. Genomic DNA microarrays can be used as probes for genomic DNA fragments of organisms obtained by separating or amplifying from each clone constituting the genomic library, respectively.

As used herein, "clone" refers to a cell line transformed with a vector comprising fragments of the entire genome of an organism. The vectors and cell lines are vectors and cell lines used in the preparation of common genomic DNA libraries, and representative vectors include Bacterial Artificial Chromosome (BAC), Human Artificial Chromosome (HAC), Transformation competent Artificial Chromosome (TAC), and YAC (Yeast Artificial Chromosome). ), Pac (P1-derived Artificial chromosome), P1 (Phage) and cosmid (but not limited to this). Examples of BAC vectors include pECBAC1 and pBeloBACII, examples of PAC vectors are pCYPAC1, examples of YAC vectors are pYAC4, examples of cosmids are SuperCos-1, and examples of P1 vectors are pAd10sacBII. The host of the vector may be appropriately selected and used according to each vector, for example, E. coli , yeast, virus. The genomic library and clones can be prepared according to conventional methods.

"Probe" referred to in the present invention is immobilized on a microarray and hybridized with homologous DNA contained in a sample, and may be DNA, RNA, cDNA or mRNA, and in the case of DNA, may be an oligomer. The probe may contain up to 85% of the repetitive sequence content in the nucleotide sequence, preferably up to 70%, more preferably up to 50%, most preferably up to 40%. The size of the probe may be 10 to 350 kb for genomic DNA, 20 to 300 bp for oligomers, preferably 20 to 100 bp, and 100 bp to 10 kb for cDNA or RNA, but is not limited thereto. It is not. The probe preferably has a nucleotide sequence comprising a single locus on the entire chromosome of the organism.

Microarray

In accordance with the present invention, a microarray may be prepared by a method comprising immobilizing a probe selected from the group consisting of RNA fragments, cDNA fragments, oligomers and genomic DNA fragments into the microarray. The microarray comprises one or two or more probes per spot, and the two or more probes immobilized on the one spot are all, a part of a chromosome site that exhibits the same gene phenotype in a chromosome or more disease It may comprise a sequence selected from the group consisting of the complement, the gene phenotype may be gene amplification or gene deletion.

The probe may be a genomic DNA fragment and may be an RNA fragment, cDNA fragment or oligomer derived from the genomic DNA fragment. Probes may be selected and used according to the purpose of the microarray. Microarray may be carried out in the same manner as a conventional microarray manufacturing method. In one embodiment, the probe is fixed to the substrate for microarray through physical bonding or chemical bonding. The substrate may be any known material, preferably a material used in a conventional microarray, and a reactor, nitrocellulose, or tertiary structure-forming material may be used to immobilize DNA on the surface of the substrate. It may be included. Examples include silicon wafers, glass, polycarbonates, membranes, polymer films such as polystyrene or polyurethanes, and porous materials.

Fixing of the probe can be carried out by a method taken in a conventional DNA microarray manufacturing method, for example, a photolithography method, a piezoelectric printing method, a micro pipetting or spotting method Can be used.

Probes may be immobilized at 2 to 100 pg per spot on a microarray, and may be immobilized by mixing one or more probes consisting of the same nucleotide sequence per spot. In addition, the spot on the microarray may be one or more times, preferably, 2 to 3 times. At this time, the spot may be circular, the diameter may be 50 to 500 um, the interval between spots may be 10 to 500 um. However, spot size and density should be appropriately adjusted according to the resolution of the microarray analysis system.

The microarray of the present invention is placed in a well of a microwell plate, such as a 96-well plate, and mechanically samples, labels, hybridizes, and washes a sample by an automatic device (eg, Biomek, Genetix robo) using an existing 96-well plate. Can be processed.

Genomic DNA fragments

Genomic DNA fragments can be obtained from the entire chromosome of an organism in a form containing the desired size and desired sequence, cloned from the organism's genomic library, or chemically synthesized.

The size of the genomic DNA fragments can be adjusted according to the analytical resolution of the genomic DNA microarray, but has an average size of 100 to 350 kb. Given that the micro-deletion, the smallest change in chromosome abnormality that occurs on the entire chromosome, is 1 Mb, the BAC chip of the present invention, which is composed of genomic DNA of 100 to 350 kb in size, can realize very high resolution when analyzing chromosomal abnormalities. It's done.

The human genomic genes mentioned in the present invention can be found in published databases such as the http://www.ncbi.nlm.nih.gov/BLAST site, in particular http://www.ncbi.nlm.nih.gov/BLAST/Blast. cgi? CMD = Web & LAYOUT = TwoWindows & AUTO_FORMAT = semiauto & aLIGNMENTS = 50 & ALIGNMENT_VIEW = Pairwise & CLIENT = web & DATABASE = nr & DESCRIPTIONS = 100 & ENTREZ_QUERY =% 28none% 29 & EXPECT = 10 & FILTER = L & FORMAT_OBJECT = Alignment & FORMAT_TYPE = HTML & NCBI_GI = on & PAGE = Nucleotides & PROGRAM = blastn & SERVICE = plain & SET_DEFAULTS.x = 34 & SET_DEFAULTS.y = 8 & SHOW_OVERVIEW = on & END_OF_HTTPGET = Yes & SHOW _LINKOUT = yes & GET_SEQUENCE = yes, each genomic DNA sequence can be identified.

Genomic DNA fragments obtained from the above known human genome libraries can be provided as probes in genomic DNA microarrays of the invention. The genomic DNA microarray includes a number of genomic DNA fragments that can all correspond to the entire genome of the organism to be diagnosed with chromosomal abnormalities, or a genomic DNA fragment corresponding to a site or a target gene for which chromosomal abnormalities are to be assayed. Include. In order to diagnose chromosomal abnormalities for the whole genome of an organism, the genomic DNA fragment number can be calculated by dividing the size of the biological genome by the size of the genomic DNA fragment. For example, for chromosomal aberration genomic DNA microarrays for humans, dividing the human genome size 3 × 10 ⁹ bp into genomic DNA fragments with an average of 100 to 350 kb requires about 30,000 to 8571 genomic DNA fragments or clones. do. The larger the size of the genomic DNA fragment, the smaller the number of genomic DNA fragments or clones required. The genomic DNA fragment comprises all of the human genome.

Genome library

Genomic libraries can be readily prepared by known methods. In one embodiment, in the case of human BAC DNA, the location and total sequence of the BAC DNA by constructing the BAC library, partial sequencing of the BAC DNA inserted into the BAC clone, and homology with the identified human genomic sequence BAC DNA can be obtained through the steps of identification, genome repositioning of BAC DNA using Fluorescent In Situ Hybridization (FISH), and BAC DNA recovery from each BAC clone. .

(1) genome library construction

The genome of the human race is isolated and then digested with restriction enzymes such as HindIII, BamHI and / or sonication, and averaged 100 to 350 Kb in size through Pulse Field Gel Electrophoresis (PFGE). Obtain a piece. It is inserted into a BAC, PAC, P1, YAC or cosmid vector treated with the same restriction enzyme and then transformed into a host cell such as E. Coli . The cell line containing each human genomic DNA fragment produced by this method is called a clone. We made a total of 96,768 clones by the above method.

(2) Confirmation of position and total sequence of inserted genomic DNA fragment

Genomic DNA fragments present in each clone are subjected to homology analysis using bioinformatics, and are subjected to homology analysis with sequences of the human genome whose base sequences are already known. The homology analysis databases available at this time are NCBI, UC Santa Cruz, and Sanger Institute.

For homology, first, end-sequencing with primers specific for the vectors transfected into the clones sequence some terminal sequences of the inserted genomic DNA fragment, eg, 100-700 bp. Since the above-described process is a state in which the base sequence and the cleavage map of the vector are confirmed, those skilled in the art can easily practice the present invention. Both terminal sequences of the analyzed genomic DNA fragments are input to a database in which the human genomic sequence is present to perform a homology search, and the position and total base sequence of the genomic DNA fragments on the human genome are identified by identifying the matching sites.

(3) Genome relocation of genomic DNA fragments using FISH

Omittable steps may be performed to reconfirm the genomic DNA fragment's position on the genome.

Genomic DNA fragments are used as probes, which are fluorescently labeled and reacted with human chromosomes in the middle stage of cell division. Fluorescent label signals are recognized to reconfirm chromosomal binding of genomic DNA fragments.

(4) recovery of genomic DNA fragments from clones

Genomic DNA fragments are recovered from each clone using known methods or commercially available genomic DNA recovery kits.

Through the above steps, the genomic DNA fragment inserted for each clone is identified. Therefore, the characteristics of genomic DNA fragments for each clone, that is, the location of chromosome, nucleotide sequence, its function, related gene or disease information are summarized, and it is an advantage that can be easily prepared by selecting appropriate genomic DNA fragment according to the use of DNA chip. There is this. In particular, when a chip for diagnosing a specific disease is desired, only genomic DNA fragments corresponding to genomic regions identified or estimated to exhibit chromosomal abnormalities during the onset or prognosis of the disease can be easily recovered from each clone.

In one embodiment of the present invention, genomic DNA fragments obtained from the BAC library for the Korean genome are shown in Table 1 below.

Preparation of Genomic DNA Microarrays for Detection of Chromosomal Abnormalities

The present invention relates to a DNA microarray for detecting chromosomal aberrations, the method for preparing the same comprising: (a) identifying genomic DNA fragments inserted into each clone from clones obtained from a genomic library of an organism, (b) chromosomal aberrations Selecting a target chromosome region to be checked, (c) selecting genomic DNA fragments capable of detecting the target chromosomal region as probes among the identified genomic DNA fragments of step (a), ( d) securing the probe to the microarray.

In the step (a), one or more genomic DNA fragments may be selected from the group consisting of human genomic DNA fragments, and preferably, suitable genomic DNA fragments may be selected depending on the target site or type of disease to be identified for chromosomal abnormalities. Can be used selectively.

In the step (b), the chromosomal abnormality may be selected from the group consisting of nucleic acid sequence variation, chromosome number amplification, chromosome number deletion, chromosomal inversion and chromosomal translocation, preferably at least chromosome number. Examples of chromosomal abnormalities include Down syndrome, Patau syndrome, Edward syndrome, Turner syndrome, Klinefelter syndrome, alpha-thalassemia retardation-16 (ATR-16) syndrome, Charcot-Marie-Tooth neuropathy 1A (CMT1A) syndrome, cat Cri-du-chat syndrome, Digeorge syndrome, Hereditary Neuropathy with Liability to Pressure palsies disease, Kallmann syndrome, Miller-Dieker syndrome, Prader-Willi Prader-willi syndrome, Rubinstein-taybi syndrome, Smith-magenes syndrome, Steroid-sulfatase gene syndrome, Williams syndrome and Wolf-Hersh Horn-hirschhorn syndrome.

In step (b), the target chromosome region is a site including disease specific variability, such as a chromosome site showing chromosome amplification or deletion specific to a disease, which can be easily adopted by those skilled in the art. Can be.

In step (c), genomic DNA fragments capable of detecting the target chromosomal site are selected. The genomic DNA fragments are then analyzed for chromosomal location, sequence, disease relevance, and function to select appropriate genomic DNA fragments. Preferably, the content of the repetitive sequence in the nucleotide sequence is at most 85%, and it is preferable to select the probe that the nucleotide sequence constituting the genomic DNA fragment has a single locus in the chromosome. Probes may comprise all, part or their complement sequences of the chromosomal site of interest.

Step (d) is a step of fixing the selected probe to the microarray substrate, which may be a conventional method. As an example, a probe may use all the DNA of a genomic DNA library vector into which a genomic DNA fragment is inserted, and the DNA fragments partially cut by the method of ultrasonic disruption of the vector DNA into which the genomic DNA fragment is inserted may be fixed to the substrate. Can be. Such a method is a conventional method for preparing genomic DNA microarrays, and therefore can be easily implemented by those skilled in the art.

The present invention, in one embodiment, exemplifies the microarray or DNA chip configuration according to the chromosomal abnormality disease.

CMT1A ( Charcot -Marie-Tooth neuropathy 1A)

CMT1A syndrome diagnoses karyotype duplication17p12 or segmental trisomy.

The DNA chip for diagnosing CMT1A syndrome may include one or more genomic DNA fragments selected from the group consisting of Nos. 1 to 235 of Table 1 or one or more oligomers of 20 to 100 bp contiguous in the fragments, or 100 bp to At least one cDNA of 10 kbp includes a fixed microarray. When two or more kinds of probes are mixed and used, the probes of each type may be mixed at the same ratio and fixed to one spot.

TABLE 1

In addition, the present invention provides a DNA microarray for diagnosing constitutional diseases. Prenatal / postnatal diagnostic DNA microarrays are intended for the detection of diseases or mutations on the whole chromosome that are targeted for normal prenatal / postnatal diagnosis, and are derived from genomic DNA or genomic DNA capable of detecting chromosomal sites associated with each disease. Oligomers, cDNAs or RNAs as probes. In this case, the prenatal / postnatal diagnostic DNA microarray may have one or more probes fixed in one array to detect chromosomal variations for each target disease. Chromosomal sites targeted for prenatal / postnatal diagnosis may be selected mainly from the group consisting of 13, 18, 21, X, and Y. Specific diseases include patau, edward, down, turner, and kleinfelter. It doesn't happen.

The genomic DNA microarray of the present invention can detect chromosomal abnormalities in a unit of at least 250 bp and has a very high resolution and is simpler than conventional karyotyping and FISH methods.

The genomic DNA microarray of the present invention has an advantage of easily obtaining a large amount of desired genomic DNA fragments according to the purpose of the genomic DNA chip from clones in which the intrachromosomal position and nucleotide sequence of the inserted genomic DNA fragments have been identified. The chromosomal abnormality on the genome can be easily detected, the chromosomal abnormality can be detected until it occurs in the intron region other than the exon region, and the chromosomal abnormality can be detected from the detected chromosomal abnormality and can be related to a specific disease. It is possible to observe the genomic variation of organisms due to human factors, to easily provide each disease-specific diagnostic chip, and is very useful for studying race-specific or human-specific polymorphism.

The genomic DNA microarrays of the present invention can diagnose diseases caused by chromosomal variations, and are particularly useful for diagnosing diseases and cancers caused by abnormal chromosome numbers.

Microarray with two or more probes fixed per spot

The present invention relates to a microarray in which two or more kinds of probes are mixed and fixed per spot. The ratio of probes mixed in one spot may be the same ratio for each kind, and the subject may be selected from the group consisting of RNA fragments, cDNA fragments, oligomers, and genomic DNA fragments. The two or more probes immobilized in the one spot comprise a sequence selected from the group consisting of all, a portion of the chromosomal region and their complements representing the same gene phenotype in chromosomal abnormalities disease, wherein the gene phenotype is amplified gene Or a gene defect. The genomic DNA may be selected from the group consisting of human genomic gene fragments, and RNA fragments, cDNA fragments or oligomers may also be derived from the genomic DNA.

Chromosome aberration detection method using microarray

The method for detecting chromosomal abnormalities of the present invention comprises preparing a microarray for detecting chromosomal abnormalities, and using a probe immobilized on the microarray, sample genomic DNA labeled with the first detectable label and standard DNA labeled with the detectable J. label. Mixed reaction at the same ratio, and measuring the hybridization ratio of each of the sample DNA and the standard DNA on the microarray, and then determining whether the sample DNA is chromosomal abnormality from the measurement result of the hybridization ratio. In the hybridization ratio, if the hybridization ratio of the sample DNA is the same as the hybridization ratio of the standard DNA, it is read as no chromosomal abnormality, and if the former is higher than the latter, it is read as having a chromosomal increase, or if the former is lower than the latter, the chromosome It can be analyzed that there is a deficiency.

The probe can be a genomic DNA fragment, oligomer, cDNA or RNA, an example of genomic DNA is the genomic DNA fragment described in Table 1.

The sample may be a biological sample obtained from a human or animal, such as body fluid (eg, saliva, blood, urine, semen, amniotic fluid, etc.), tissue, or cell, and DNA may be extracted and separated from the biological sample by a conventional method. have.

The first label and the second label may be the same or different materials, for example, independently of each other may be a fluorescent material, radioisotope, chemiluminescent or enzyme. Examples include Cy3, Cy5, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 658, Cyanine-3, Cyanine-5, Fluorescein ( reactive dyes, amounts with fluorescein, bodipy, Texas red, Fluorescein Isothiocyanate (FITC), rhodamine, d-NTP (including d-UTP), amino-allyl modified dNTPs Horseradish peroxidase and biotin.

This will be described in more detail as follows.

Step 1: Extract the Sample (Genome DNA)

Genomic DNA is extracted from the sample using conventional methods or known kits. Sample genomic DNA is prepared from 100 ng to 1 ug.

Step 2: Cover

Samples and standard genomic DNA are labeled with different labels, respectively, through random priming, and then labeled samples and standard genomic DNA are obtained.

In one example, the sample is labeled with Cy3 (blue) and the standard genomic DNA is labeled with Cy5 (red).

Step 3: Reaction to Microarray Chips

Labeled specimens and standard genomic DNA are mixed in a 1: 1 weight ratio, and the mixture is treated by microarray, reacted and washed.

The amount of DNA of the mixture to be treated in the microarray may be 200 to 2 ug, and upon treatment, the mixture may be treated by suspending in hybridization buffer. Hybridization buffers include known solutions. In addition, the BAC chip can react with a material that can prevent non-specific hybridization, such as salmon sperm DNA, before the mixture is processed.

Step 4: Analyze

After completion of the reaction, the microarray is scanned in a detectable scanner of a labeling material, and then the hybridization is analyzed using a program in which the probe information and the spot location on the microarray are input.

When the chromosome of the sample is normal, the spot is yellow because the ratio of the sample hybridized to the probe and the standard genomic DNA is 1: 1.

If the chromosome of the sample has an amplified variation, the probe hybridizes to the probe at a higher rate than the standard genomic DNA, so that the spots are blue.

If the chromosome of the sample has a deletion, the probe hybridizes at a high rate of standard genomic DNA to the sample, so that the spots appear red.

Therefore, by confirming the fluorescence of each spot on the microarray, it is possible to detect amplification or deletion mutation of specific DNA of the sample.

Chromosomal abnormality detection method specific to disease

The present invention also relates to a method for detecting a chromosomal abnormality specific to a disease, comprising the steps of: (a) identifying each genomic DNA fragment inserted into the clone from each clone obtained from the genome library of the organism, (b) the ( a) immobilizing all or a portion of the genomic DNA fragments identified in step a) into a microarray; (c) detecting the sample genomic DNA labeled with the detectable primary label of a diseased patient on the microarray probe; Mixing and reacting the standard DNA labeled with a possible second marker at the same ratio, measuring the hybridization ratio of the sample DNA and the standard DNA, and (d) analyzing the hybridization ratio to determine the site of the chromosomal abnormality of the diseased patient, and Identifying the chromosomal aberration form.

By the above method, the disease-specific chromosomal abnormalities and abnormalities of each disease can be easily identified if the disease-specific chromosomal abnormalities are confirmed.

In one embodiment, BAC chips are prepared using BAC clones obtained from the BAC library of the human genome. In this case, the BAC DNA constituting the BAC chip may correspond to the entire human genome or may be for a specific chromosome or gene.

BAC chips composed of BAC DNA corresponding to the entire human genome can be used for the following purposes.

(1) The detection of chromosomal abnormalities in the entire genome of a sample is used to diagnose the disease when the health status of the sample and the detected chromosomal abnormalities are linked to a specific disease.

(2) By analyzing the DNA of a specific diseased patient as a sample, the chromosomal abnormality associated with the specific disease is identified and used as a useful indicator for later diagnosis of the same disease.

(3) Analyze chromosomal abnormalities caused by certain substances, such as drugs or compounds.

(4) Identify genomic diversity by individual or race.

BAC chips for specific chromosomes or genes can be used to more precisely detect chromosomal abnormalities for targeted sites.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1 Preparation of BAC Clone

1-1. Genomic library manufacturing

After separating Korean genome, DNA fragment of about 100Kb was obtained through HindIII enzyme treatment and PFGE. After ligation of the average 100 Kb DNA fragment into the BAC vector, E. Coli. The host cell was transformed. Where each of the DNA fragments contains E. Coli . The cell line is called a clone, and a total of 96,768 clones were obtained by constructing the genome library (FIG. 1).

1-1-1. Genome isolation

Total genomic DNA was extracted from 20 ml of Korean semen and subjected to electrophoresis on agarose gel to analyze the quality and quantity of the extracted DNA.

1-1-2. Fragmentation and Purification of the Genome

Genomic DNA was digested with BamHI and electrophoresed on a PFG gel to separate DNA fragments by taking only the portion developed at the 100 Kb position.

1-1-3. Transformation

DNA fragments were inserted into BamHI of BAC vector (pECBAC1), and then transformed into host cells (Competent cell, E. coli DH10B BAC). After incubation in a solid medium to obtain each clone was inoculated in a 96 well format cell culture blot again and incubated at 300 rpm for 18 hours in a 37 ℃ rotary incubator.

1-1-4. Library Cell Stock Preparation

Divide 25 μl of 65% glycerol into 384 well plates and add 25 μl of cells cultured in 96 well culture blots (cells cultured in four 96 well format cell culture blots are collected and stored in one 384 well plate). . The 384 wells were sealed at -70 ° C with the top sealed.

1-2. End-sequencing

500bp of both ends of the 100Kb DNA fragment was analyzed with a specific primer to the BAC vector to confirm and record the inserted genomic DNA information for each clone.

1-2-1. Isolation of BAC DNA (Mini-prep.)

Put 1 ml of 1 X LB into a 96 well culture blot and inoculate 2 µl of the library cell stock solution. At this time, cells stored in one 348 well plate were cultured by dividing into four 96 well culture blots. Incubated at 37 ° C., 18 hours, and 300 rpm using a rotary incubator, and then sample stock solution was prepared at 5 × with 25 μl per well. The remainder was centrifuged (1000 rpm, 15 minutes) to obtain cells. Montage mini-prep. DNA was recovered using the kit.

1-2-2. Sequencing

Both ends of genomic DNA fragments inserted into each clone were analyzed using T7 and M13R primers, and the sequences were read with an ABL 3700 automated sequencer.

1-3. Bioinformatics

The analyzed sequences were compared to homology with the entire human genome sequence found in the Human Genome Project using bioinformatics techniques. Through this process, the genome location and total sequence of the BAC DNA contained in each clone were revealed.

1-3-1. Blast search

Blast search was performed using the analyzed terminal sequences, and homology analysis confirmed the location on the genome of each clone.

1-3-2. Gene / Marker Screening

Of the clone-specific genomic DNA fragments obtained from the genomic library, clones containing cancer-related genes / STS markers were selected.

1-4. BAC Clone mapping by FISH

In order to more accurately identify the genomic location of each BAC clone, FISH was performed using the genomic DNA fragment contained in each BAC clone as a probe. The FISH technique uses the principle of complementary binding of DNA to identify the position of the probe in the chromosome by directly binding to a chromosome immobilized on a glass slide with a probe that specifically binds to a specific site of the chromosome.

Chromosome positions of the identified BAC clone-specific genomic DNA fragments are shown in Table 1.

Example 2: Fabrication of BAC Chips

2-1. Selection of clones to be used for the fabrication of BAC chips

For the purpose of diagnosing specific genetic diseases associated with abnormal numbers of human chromosomes, 1440 clones were selected for each chromosome number in the BAC library. 1440 clones are shown in Table 2.

Table 2

Chromosomal location and total size of each of the selected 1440 genomic DNA fragments are shown in Table 3 below and FIGS. 2A-H.

Table 3

Chromosome number Size (Mbp) Genomic DNA Fragment Numbers Used on BAC Chips Genomic NA Fragment Size (Mbp) Chromosome counterpart (%) One 246 103 0.1 4.2 2 243 110 0.1 4.5 3 199 92 0.1 4.6 4 191 64 0.1 3.4 5 181 73 0.1 4.0 6 170 81 0.1 4.8 7 158 83 0.1 5.3 8 146 52 0.1 3.6 9 136 66 0.1 4.9 10 135 68 0.1 5.0 11 134 73 0.1 5.4 12 132 56 0.1 4.1 13 113 42 0.1 3.7 14 105 43 0.1 4.1 15 100 39 0.1 3.9 16 90 36 0.1 4.0 17 81 57 0.1 7.0 18 76 33 0.1 4.3 19 63 38 0.1 6.0 20 63 39 0.1 6.2 21 46 40 0.1 8.7 22 49 50 0.1 10.2 X 153 92 0.1 6.0 Y 50 17 0.1 3.4 total 3060 1440 0.1 (mean) 4.7 (mean)

2-2. Isolate genomic DNA fragments from clones

2 µl of each BAC clone stock solution was inoculated into 85 ml of 1X LB (DIFCO, Cat. No 244620) containing 400 µl of chloramphenicol solution (100 ml of 100% alcohol + 340 mg of chloramphenicol). After incubating at 250 rpm for 15 hours at 37 ° C, pellets were obtained by centrifugation at 6,000 rpm, 4 ° C, and 5 minutes, and DNA was extracted using QUIAGEN-tip 100 (QIAGEN plasmid midi kit).

850 µl of 1 ml of BAC DNA was sonicated for slide drip DNA and 3 µl for electrophoresis on agarose gel. In addition, 20 μl of randomly selected BAC DNA was used for pulse field gel electrophoresis (PFG) electrophoresis.

2-2-1. Agarose Gel Electrophoresis

In order to confirm the isolated BAC DNA, 3 μl of BAC DNA dissolved in distilled water was taken, reacted with Not I restriction enzyme treatment, and developed on 0.85% agarose gel. The development pattern was confirmed to select BAC DNA containing genomic DNA fragments.

2-2-2. PFG electrophoresis (CHEFF 162-00133, BIO-RAD)

Of the 48 BAC clones, a single BAC DNA extraction unit, 6 randomly selected PFG electrophoresis was performed, and if any of the 6 clones were defective, 48 BAC clones, which were once extracted, were extracted again.

6-3. Spotting

Genomic DNA fragments obtained from 1440 clones were dropped into microarrays. In this experiment, 12 subarrays of FIG. 3 were used, and each subarray had 360 (30 × 12) spots, and the same genomic DNA fragment was dropped three times in the horizontal direction. That is, 120 (360/3) different genomic DNA fragments are stored in one subarray, and 1440 genomic DNA fragments are present in 12 subarrays. The number of genomic DNA fragments and target chromosome numbers for each subarray are shown in Table 4 below and FIGS. 4A-F.

Table 4

Chromosome number Subarray number One 2 3 4 5 6 7 8 9 10 11 12 sum One 9 6 11 11 5 5 10 13 9 7 9 8 103 2 9 11 9 8 4 5 13 5 11 9 13 12 109 3 5 6 5 10 10 12 11 8 7 7 8 3 92 4 4 7 3 3 3 4 6 6 6 8 8 5 63 5 2 4 7 10 7 5 7 3 7 10 8 3 73 6 7 5 12 6 5 7 5 5 8 9 7 5 81 7 9 8 5 7 5 8 8 7 5 10 5 6 84 8 6 3 5 2 3 4 5 7 4 5 6 3 53 9 4 0 6 5 One 3 8 8 8 5 6 2 56 10 6 8 4 2 7 8 3 5 7 10 5 3 68 11 7 4 9 5 8 11 4 5 4 3 6 7 72 12 6 One 4 4 5 5 7 8 3 6 4 4 57 13 2 One 2 5 2 2 2 6 7 3 6 4 42 14 5 3 2 2 One 7 4 3 6 3 3 5 44 15 4 One 2 2 4 5 3 4 6 3 4 2 40 16 0 2 One 4 2 2 One 5 5 3 2 9 36 17 5 9 5 4 5 2 One 5 2 3 5 11 57 18 One 3 4 2 3 3 5 3 4 One 2 2 33 19 One 5 One 6 4 7 One One One 2 One 8 38 20 4 9 3 3 4 2 One One 4 2 One 5 39 21 5 6 5 4 7 One One 0 3 0 4 4 40 22 7 5 5 7 13 6 2 0 2 2 0 One 50 X 7 8 4 3 7 3 6 6 0 5 5 4 58 Y One 2 0 4 2 0 3 2 One 0 0 2 17 sum 120 120 120 120 120 120 120 120 120 120 120 120 1440

2-3-1. Sample Preparation

After centrifugation (12,000 rpm, 10 min) of the BAC DNA solution, 850 µl of the supernatant was collected, and subjected to sonication under the following conditions.

Ultrasonic Decomposition Conditions

Time: 5 seconds, temperature: 25 ° C, pulse: 0.1 / 0.1

All of the 1440 BAC DNAs that had been sonicated were electrophoresed on 1% agarose gel to confirm that sonication was successful. Sonicated DNA should be distributed in the 0.5Kb to 10Kb position. The BAC DNA solution was concentrated / dried in SpeedVAC (SAVANT, AES2010-220), dissolved in 45 μl of distilled water, and mixed to confirm viscosity. The highly viscous sample was repeatedly subjected to ultrasonic decomposition. Then 45 μl of the drop solution (100% DMSO, 1.2 ug / μl nitrocellulose) is added and transferred to 384 plates (30 μl per well). Fill in the blanks with 30 μl of 50% DMSO.

The prepared sample is stored at -70 ° C when stored for at least one month, and stored at -20 ° C when stored for less than one month. Defrost before use and spin down for 1 minute at 2000 rpm.

2-3-2. Board Preparation

A glass substrate of 25 mm x 75 mm x 1 mm size was prepared. The glass substrate is amino-silane treated.

2-3-3. Drip

The GeneMachine MicroGrid100 was used to instill at 5-10 pg per spot of the substrate. At this time, the nitrogen cylinder value was 103.4 kPa to 137.9 kPa, and the vacuum pump was operated to maintain 74.5 kPa to 101.6 kPa.

Experimental Example 1 Analysis of Chromosome Abnormalities

Each sample cell was obtained from patients with Down's syndrome, Edward's syndrome, Patau's syndrome, Turner syndrome and Kleinfelter's syndrome, which were responded to the BAC chip of Example 2.

(Experimental method)

1. Extraction of Genomic DNA

500 ng of sample DNA was obtained using a commercially available Puregene DNA isolation Kit (Gentra, Cat. No. D5500A) from blood. In addition, a standard sample was obtained by performing the same method on reference cells.

2. Probe Markers

Samples and standard samples were each labeled with Cy3 (Cy3-dCTP) and Cy5 (Cy5-dCTP) by random priming, and then purified by QIAGEN purification kit.

1) Cover

(1) Take 21ul (500ng) of DNA (sample or standard sample) into the tube and add 20ul of 2.5 X random reaction buffer.

(2) The tube is boiled for 5 minutes in a 100 ° C heating block, and then immediately left on ice for 5 minutes.

(3) After spindown, add 2 mM dNTP solution to the tube by 5ul. (2mM dNTP solution preparation: 100mMdATP, dGTP, dTTP each 20ul, dCTP 10 each and add 30ul of TE and mix well)

(4) Add 3 μL of Cy3 for the sample and 3 Cy5 for the standard sample.

(5) Add 1.2ul of klenow fragments.

(6) After pipetting and evenly mixed, spin down to react in a 37 ℃ incubator.

2) Purification using spin column (Qiagen, Qiaquick PCR purification kit)

(1) Add 50ul of PB (Binding Buffer) to 50ul of the reaction solution and drop it into the prepared spin column.

(2) Centrifuge the spin column at 13,200 rpm for 1 minute 30 seconds.

(3) Put 750ul PE (washing buffer) into the spin column.

(4) Centrifuge the spin column at 13,200 rpm for 1 minute 30 seconds.

(5) Discard the solution collected in a 2 ml tube.

(6) Centrifuge.

(7) Drop 50ul of EB (Spill Buffer) into the column and let it stand at room temperature for 3 minutes.

(8) Centrifuge the spin column at 13,200 rpm for 1 minute 30 seconds.

(9) Drop 30 ul of EB into the column and leave at room temperature for 3 minutes.

(10) Centrifuge the spin column at 13,200 rpm for 1 minute 30 seconds

3) ethanol precipitation

(1) 80ul of sample DNA (Cy3 labeling), 80ul of control DNA (Cy5 labeling), 30ul of Cot I DNA, 20ul of 3M sodium citrate, and 500ul of chilled 100% ethanol.

(2) Leave at -20 ° C for 60 minutes.

(3) Centrifuge at 13,000 rpm for 4 minutes at 4 ° C.

(4) After centrifugation, a purple precipitate is formed. Discard the supernatant.

(5) After mixing 500ul of cold 70% ethanol, centrifuge for 10 minutes at 13,000rpm, 4 ℃.

(6) Discard the supernatant and spin down to remove the remaining ethanol with a pipette.

(7) Leave in the dark for 10 minutes.

(8) Add 44ul of array hybridization solution and 4ul of yeast tRNA solution to the precipitate. If left in the dark for more than 30 minutes, the precipitate melts to a purple color and is used as a probe sample solution.

4. Hybridization reaction

-BAC Chip Pretreatment-

(1) Mark the top and bottom of the BAC chip with a diamond pen.

(2) Add 10 uL of salmon sperm DNA to 30 uL of the hybrid solution.

(3) The mixture is denatured for 10 minutes on a 70 ° C heat block and placed on ice for 5 minutes.

(4) Load 40 uL of the mixture on the BAC chip and place in a damp slide box for 30 minutes.

(5) BAC chip is immersed in 3 distilled water for 10 seconds and washed twice.

(6) The BAC chip is washed with isopropanol and then centrifuged at 550 rpm for 5 minutes to dry the slides.

Hybridization

(1) 44 ul of purple probe sample solution is denatured on a 70 ° C. heat block for 15 minutes and then reacted with a BAC chip.

(2) It is left to stand in a 37 degreeC incubator for 1 hour.

(3) Load the hybridization solution.

(4) Cover glass is covered with 20 X 40mm.

(5) Place in a slide box and place on a shaker at about 5 rpm. Hybridize for 48-72 hours in an incubator maintained at 37 ° C and 100% humidity.

-wash-

(1) A solution containing 50% formamide and 2X SSC (pH7.3) was added at 46 ° C to wash the BAC chips for 15 minutes.

(2) Wash for 30 min with a solution at 46 ° C. containing 2 × SSC and 0.1% SDS.

(3) 100 mL of a mixed solution (pH 8.0) containing PN buffer (0.2 M Na 2 HPO 4 (pH 9.0) and 0.2 M NaH 2 PO 4 (pH 4.3), 100 mL of distilled water, and 200 μL of NP40) were washed at room temperature for 15 minutes.

(4) Wash at room temperature for 5 minutes with 2X SSC.

(5) Wash sequentially with 70%, 85% and 100% ethanol at room temperature for 1 minute.

(6) The slides are dried by centrifugation at 550 rpm for 5 minutes.

4. Scanning and Analysis

The BAC chip was inserted into the microarray chip analyzer (MAC VIEWER, Class 2, A661200) and scanned, and each scanned image was obtained through Cy3 and Cy5 filters. The scanned image results were analyzed using a BAC chip analysis program (Macrogen) in which information about genomic DNA fragments deposited on the BAC chip was input.

1) Scanning

end. Preparation before Use of Microarray Chip Analysis System

Connect the microarray chip analyzer to a PC with PCI. The recommended specifications of the PC are as follows:

-IBM Pentium III or later

Windows NT 4.0 or Windows 2000

-768 MB RAM or more

-30 GB or more hard disk

-Save / Restore CD-ROM

1280 x 1024 display system (16M colors)

Upon scanning, the channels were selected as cy3 and cy5 and the exposure time was used 1 sec. Scanned after focusing, and file name was saved.

2) analysis

The scan file was run in BAC Chip Analysis Program (Macrogen), an analysis software in which information on the BAC chip was input, to obtain a log ratio chart.

The final experimental results of the BAC chip show the log2 base T / R values for the 1440 BAC clones that contain the entire autosomal (Nos. 1-22) and even X and Y sex chromosomes in chromosome order. The T / R value is obtained by dividing the fluorescence intensity (CY3) of the test by the fluorescence intensity (CY5) of the reference.It is 0 when the two fluorescent dyes have the same intensity, and 0 when the fluorescence intensity of the sample is strong. > 0.2, and if the intensity of fluorescence of the control group is strong, 0 <0.2. Determine the numerical change of each chromosome from the average (chromo-mean) and chromo-mean graph of the log2 base T / R values of each chromosome depending on whether each chromosome is amplified or lost It was. In the case of autosomal bodies, the number of chromosomes in the sample and the control group is the same, and the same fluorescence intensity is obtained. Therefore, the average chromo-mean of each chromosome converges to zero. Will increase. The sex chromosome showed the same pattern as the autosomal chromosome according to the number of chromosomes. The control group uses male DNA, so if the sample is male DNA, it is determined by the same method as the autosomal one. If the sample is female DNA, the average value of Y chromosome is less than -0.4, and the X chromosome is about 0.2 or more. Increases. The numerical abnormality of each chromosome is determined by Table 5 below.

Table 5

chromosome Kayotype Chromosome log2T / R mean (M)  Chromosome Nos. 1 to 22 Diploid -0.2 <M <0.2 deletion (Monosomy) M <-0.2 Amplification (Trisomy) M> 0.2   X chromosome Male (XY) -0.2 <M <0.2 Male, Duploid (XXY) M> 0.2 Female (XX) M> 0.2 Female, Deletion (Monosomy, XO) -0.2 <M <0.2  Y chromosome Male (XY) -0.4 <M <0.4 Male, Duploid (XXY) M> 0.4 Female (XX) M <-0.4

(result)

5 to 14 show the results of diagnosis of abnormalities in the number of chromosomes for the sample DNA. FIG. 5 shows that the log2 T / R value of the X chromosome is 0.421, which is greater than 0.2, so that the sex of the sample is female, and the log2 T / R value of the autosomal excluding the chromosome 13 is between -0.2 and 0.2. It can be seen that it belongs to normal, but chromosome 13 is 0.273, so it can be seen that the 13th chromosome was amplified. That is, the results of FIG. 5 indicate that the patient is Pattau syndrome.

In Fig. 6, the log2 T / R value of chromosome 13 is 0.32, the log2 T / R value of the X chromosome is 0.011, and the log2 T / R value of the Y chromosome is -0.076, so it is diagnosed as a man with Patau syndrome.

Fig. 7 shows that the log2 T / R value of chromosome 18 is 0.390, the log2 T / R value of the X chromosome is -0.004, and the log2 T / R value of the Y chromosome is -0.118.

Fig. 8 shows that the log2 T / R value of chromosome 18 is 0.367, the log2 T / R value of the X chromosome is 0.380, and the log2 T / R value of the Y chromosome is -1.227.

9 is diagnosed as a woman with Down syndrome because the log2 T / R value of the chromosome 21 is 0.263, the log2 T / R value of the X chromosome is 0.428 and the log2 T / R value of the Y chromosome is -1.186.

10 shows that the log2 T / R value of chromosome 21 is 0.260, the log2 T / R value of the X chromosome is 0.344, and the log2 T / R value of the Y chromosome is -0.149. And the X chromosome is diagnosed with one more amplified karyotype XXY.

11 is diagnosed as Turner syndrome with karyotype XO since the log2 T / R value of the X chromosome is -0.071 and the log2 T / R value of the Y chromosome is -1.987.

Fig. 12 shows diagnosis of Klinefelter syndrome with karyotype XXY since the log2 T / R value of the X chromosome is 0.446 and the log2 T / R value of the Y chromosome is -0.0091.

FIG. 13 shows that the log2 T / R value of the X chromosome is 0.395, and the log2 T / R value of the Y chromosome is −1.131.

14 is diagnosed as a normal male since the log2 T / R value of the X chromosome is -0.010 and the log2 T / R value of the Y chromosome is -0.108.

Experimental Example 2

The experiment was carried out in the same manner as in Experimental Example 1, except that both the sample and the control DNA were labeled with Cye3. In the post-scan analysis, the log2 T / R value of the spot signal was used and analyzed according to the criteria of Table 5. Figure 15 shows the results of BAC chip analysis of Down syndrome sample DNA, a signal amplified chromosome 21 was observed.

As described above, the chromosomal abnormality detection method of the present invention enables a quick and accurate assay of chromosomal abnormalities to easily diagnose the state of health and disease of a specimen. In addition, the method of the present invention may identify disease specific chromosomal abnormalities and provide an indicator for diagnosing disease.

Claims (10)

(a) at least one genomic DNA fragment selected from the group consisting of 1 to 235 in Table 1; (b) 20-100 bp of oligonucleotides located consecutively within the genomic DNA fragment; (c) 100 bp to 10 kbp of cDNA derived from said genomic DNA fragment; And (d) complements of said genomic DNA fragments, oligonucleotides and cDNAs At least one probe selected from the group consisting of The probe has a maximum sequence repeat rate of 85% in the nucleotide sequence, The base sequence constituting the probe has a single locus in the chromosome, Charcot-Marie-Tooth neuropathy 1A (CMT1A) diagnostic microarray: TABLE 1

The method of claim 1, wherein the genomic DNA fragment is a BAC (Bacterial Artificial Chromosome), HAC (Human Artificial Chromosome), TAC (Transformation competent Artificial Chromosome), YAC (Yeast Artificial Chromosome), PAC (P1-derived Artificial Chromosome) , Prepared by inserting into a vector selected from the group consisting of P1 (phage) and cosmid. The microarray of claim 2, wherein the probe is prepared by inserting the genomic DNA fragment into a BAC (Bacterial Artificial Chromosome). The microarray of claim 1, wherein the probe is fixed at a concentration of 2 to 100 pg per spot. The microarray of claim 1, wherein at least one probe is fixed to one spot. The microarray of claim 1, wherein at least two probes are fixed per spot. A microarray according to any one of claims 1 to 6 and a labeling means detecting means, A sample genomic DNA labeled with a detectable first label and a standard DNA labeled with a detectable second label are mixed and reacted at the same ratio with the probe immobilized on the microarray, and the hybridization ratio of each of the sample DNA and the standard DNA is It is characterized in that by determining the chromosomal abnormality of the sample DNA from the results Charcot-Marie-Tooth neuropathy 1A (CMT1A) diagnostic kit. The kit of claim 7, wherein the first label and the second label are different materials or the same material selected from the group consisting of radioisotopes, fluorescent materials, chemiluminescent materials and enzymes. The method of claim 7, wherein The sample is a body fluid, tissue or cell, The body fluid is selected from the group consisting of saliva, blood, urine, semen and amniotic fluid. The method of claim 7, wherein If the hybridization rate of the sample DNA is the same as the hybridization rate of the standard DNA, it is read as no chromosome abnormality. If the hybridization rate of the sample DNA is higher than the hybridization rate of the standard DNA, read that there is an increase in chromosome, When the hybridization ratio of the sample DNA is lower than the hybridization ratio of the standard DNA, the kit, characterized in that the chromosomal defect is read to determine the chromosomal abnormality of the sample DNA.

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