[Invention Title] METHOD FOR MEASURING THE CHROMOSOME, GENE OR NUCLEOTIDE SEQUENCE COPY NUMBERS USING CO- AMPLIFICATION OF ARTIFICIAL SNP SEQUENCES

[Technical Field] The present invention relates to a method of measuring the copy number of a chromosome, gene or specific nucleotide sequence. In addition, the present invention relates to a kit for analyzing the number of the chromosome, gene or specific nucleotide sequence. More specifically, the invention relates to a method of measuring the copy number of the chromosome, gene or specific nucleotide using an artificial single-nucleotide polymorphism (SNP) sequence. In particular, the invention relates to a method of co-amplifying genomic DNA sequences and artificial SNP sequences and then measuring the copy number change of the chromosome, gene or specific nucleotide sequence due to deletion, duplication and the like of the chromosome through a relative amount of the amplified genes.

[Background Art] Changes in specific chromosomal sequences are frequently implicated in human diseases and syndromes. Such changes include the addition of one entire chromosome or the deletion of one entire chromosome as in Down's syndrome, deletions of several million base pairs as in DiGeorge syndrome and deletions or duplications of small chromosomal fragments as in Becker or Duchenne muscular dystrophy. A subtelomeric deletion is also frequently reported in mental retardation patients (Lamb et al., 1989). In addition, chromosomal regions of specific genes such as BRCAl or MLH1IMLH2 are commonly changed in tumors, which is known to be important for gene expressions (Petrij-Bosch et al., 1997; Wijnen et al., 1998). An analysis of the copy number change of genes can be important for the treatment of the cancer patients, as can be seen from an example of using ERBB2-specific antibodies to treat a breast cancer patient having ERBB2 gene amplified (Leyland- Jones and Smith, 2001). At present, many techniques are used to determine the copy number of chromosomal changes. The most standardized method of measuring the number and structural changes of the chromosomes is a karyotyping method. According to this method, it is required to culture the patient's blood, fibroblast or amniotic cells, and much time and manpower are necessary to interpret the result thereof. The karyotyping method usually can detect 1 mega base or more of the chromosomal changes only. This sensitivity issue can be made up for with a fluorescent in situ hybridization (FISH) method. However, the FISH method also requires much time and manpower and does not usually measure the changes of four or more different target genes at a time (Klinger et al., 1992). In addition, a multicolor chromosome painting method is introduced as a method for automatization of the karyotyping. The method allows the deletion, duplication or translocation of the chromosome to be easily detected by labeling portions of each chromosome with fluorescent materials of different colors (U.S. Patent No. 6,066,459). Although the multicolor chromosome painting method increases the sensitivity somewhat, compared to the karyotyping method, it basically needs a cell culture and a post-process required for the karyotyping. In order to overcome the requirements of time and manpower, several molecular methods have been recently developed to detect the chromosomal changes. Array based-comparative genomic hybridization (CGH) is one of the most promising methods and there have been attempted many trials for application to the diagnosis on genetic diseases or the detection of chromosomal changes in cancer tissues (Pinkel et al, 1998; U.S. Patent Nos. 6,197,501 and 6,159,685). This method immobilizes BAC clones on a substrate surface to form an array, and pre-labeled standard DNA and sample DNA are hybridized to the array. According to the method, a relative amount of signals from the standard and sample DNAs is compared to detect the chromosomal changes such as deletion or duplication. In addition, there is a method of determining the copy number by measuring the relative amplification with multiplex PCR method (Rahil et al., 2002). As a modified method thereof, it is recently introduced a multiplex ligation-dependent probe amplification (MLPA) (Schouten et al., 2002; Carrino, 1996). The relative amplification degree is relatively stabilized during the amplification of genes with the MLPA method. However, a standard deviation is still large (standard deviation = 0.13-0.3), so that the method has been not developed to a satisfactory level for the diagnosis of chromosomal abnormality patient. In addition, there is a multiplex amplifiable probe hybridization (MAPH) method of hybridizing short probes with genomic DNA, collecting and amplifying them quantitatively, thereby determining the copy number (John A. L. Amrmour at al., Nucleic Acids Research, 2000, Vol. 28, No. 2). However, since the method also has a still large Standard deviation, it is difficult to apply the method to the diagnosis of chromosomal abnormality patient. Loss of heterozygosity (LOH) is the most common method to detect deletion or duplication of chromosome, gene or specific nucleotide sequence. However, the LOH method requires a standard sample having the same allele constitution as that of the remaining region of a sample except the deleted or duplicated region. Accordingly, the method is mostly used to diagnose the chromosomal abnormality in the cancer tissues using a non-cancer tissue as the standard sample. For a research of the LOH, a microsatellite marker (Call et al., 1990) or a SNP (Lindblad-Toh at al., 2000) may be used. The LOH method cannot distinguish whether a chromosomal change is deletion or duplication, except the homozygous deletion. Pont-Kindon and Lyon (2003) reported another method of using the SNP to detect the chromosomal abnormality. They used a melting curve analysis to detect the relative amount of heterozygous alleles. The method detects that there is a trisomy when the relative amount of two alleles is different from a normal ratio. This method cannot alse distinguish whether a chromosomal change is deletion or duplication of the chromosome, either. In addition, since the method needs at least one or more heterozygous alleles in a specific locus so as to detect a copy number change, and it is required to include at least 5-6 SNP sites for the assay.

[Disclosure] [Technical Problem] Accordingly, the present invention has been made to solve the above problems. An object of the present invention is to provide a method capable of allowing more accurate values to be obtained compared to the other molecular methods of determining the copy number of a specific gene and thus accurately measuring the copy number change of a chromosome, gene or specific nucleotide sequence, including duplication or deletion of chromosome as well as capable of remarkably reducing costs and required manpower, when measuring the copy number of the chromosome, gene or specific nucleotide sequence.

[Technical Solution] In order to achieve the above object, there is provided a kit for analyzing the copy number of a chromosome, gene or specific nucleotide sequence using amplification, the kit comprising: one or more artificial single nucleotide polymorphism (SNP) sequences including one or more artificial SNPs having a base change introduced artificially, the artificial SNP sequences to be amplified including a primer sequence part are same as each of the corresponding wild-type genomic DNA sequences, except the artificial SNPs; amplification means for amplifying at least a part of both the artificial SNP sequence and a test sample DNA sequence for which the copy number of chromosome, gene or nucleotide corresponding to the artificial SNP sequence is analyzed, the amplification means amplifying the sequences themselves or signals of the sequences; a primer consisting of sequences existing in common in the test sample DNA sequences and the artificial SNP sequences; means for distinguishing the amplified test sample DNA sequences from the amplified artificial SNP sequences, the means detecting a base, different from the wild-type genomic DNA sequence existing in the artificial SNP sequence to distinguish the amplified test sample DNA sequence from the amplified artificial SNP sequence and thus to measure each gene amounts of the genomic DNA sequences (A, B, C, D....) of the test sample and the artificial SNP sequences (A', B', C5 D' ....); and a data-storing means for calculating a ratio (A/ A', B/B', CIC, D/D') of a gene amount (A', B', C, D'....) of the amplified artificial SNP sequences and a gene amount (A, B, C, D....) of the corresponding amplified test sample DNA and storing the gene amount data (A/A, B/B', C/C, D/D') of aSNP normalized test sample obtained from the calculation, so as to normalize the measured gene amount of the test sample DNA sequences based on the artificial SNP sequences, the means further storing data [RRAB = (A/A')/(B/B!), RRAC = (A/A)/(C/C), RRAD = (A/A')/(D/D'), RRBC = (B/B')/(C/C)....], which is data of comparing the gene amount data of the respective aSNP normalized test sample for each of the gene species with those of the remaining gene species, the means further co-amplifying standard sample DNA sequences (A*, B*, C*, D*....) and the artificial SNP sequences (A, B', C, D'....) to convert a gene amount of the amplified standard sample DNA sequences into data of aSNP normalized standard sample (AVA', BVB', CVC, DVD'....) and then to convert the converted gene amount data (AVA, BVB', CVC, DVD'....) into ratio data with other genes [RRA*B* = (AVA')/(BVB')5 RRA*C* = (AVA')/(CVC), RRA*D* = (AVA')/(DVD')5 RRB*c* = (BVB')/(CVC)....] to store them, and the means further storing a ratio [nRRΛB = RRAB/ RRA*BS nRRAc = RRAC/ RRA*C*, nRRAD = RRW RRA*D*, ΠRRBC = RRBC/ RRB*CV - . -] of the gene amount data of the aSNP normalized test sample and the gene amount data of the aSNP normalized standard sample for each of the genes. According to an embodiment of the invention, the artificial SNP sequence has preferably a base change that occurs less than 1% in the natural world, and more preferably a base change of less than 0.1% that is not reported yet. When the base change, which occurs 1% or more naturally, is introduced, it is difficult to achieve the object of the invention because the base change cannot be used so as to distinguish the genomic DNA sequence from the artificial SNP sequence. A sample having the rare base change may cause an abnormal result. In this case, when the test is done with the DNA sample only without spiking the artificial SNP sequence, the base change in DNA sample can be detected, thus it can be validated that the ratio change is not caused by a change of the copy number in the sample DNA. According to another aspect of the invention, there is provided a method of measuring the copy number of a chromosome, gene or specific nucleotide sequence comprising: a first step of competitively amplifying one or more artificial single nucleotide polymorphism (SNP) sequences including one or more artificial SNP having a base change introduced artificially and specific nucleotide sequences of one or more test sample DNA sequences for which copy number is measured, the artificial SNP sequences are same as respective corresponding wild-type genomic DNA sequences, including a primer sequence part, except the artificial SNPs; a second step of distinguishing the amplified test sample DNA sequence from the corresponding amplified artificial SNP sequence to measure each amplification amount of both the sequences; and a third step of determining the copy number of the chromosome, gene or specific nucleotide sequence, based on the measured amplification amount.

[Advantageous Effects] According to the invention, it is possible to obtain more accurate value than the other molecular methods determining the copy number of the specific genes as well as to remarkably reduce the time and necessary manpower, when analyzing the change of the chromosome.

[Description of Drawings ]

The above and other objects, features and advantages of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. 1 is a schematic view illustrating artificial SNP sequences (A' and B') and wild-type sequences (A and B); FIG. 2 is a schematic view showing a method of measuring the copy cumber of a chromosome, gene or specific nucleotide sequence using SNP, according to an embodiment of the invention; FIG. 3 is a schematic view illustrating results obtained from a standard sample and a test sample (aneuploidy sample); FIG. 4 is a schematic view showing a single base extension method among the methods of distinguishing amplified products from the genomic DNA sequence and the artificial DNA sequence; FIG. 5 is a schematic view showing another method of distinguishing amplified products from the genomic DNA sequence and the artificial DNA sequence; FIG. 6 is a schematic view showing a method of using a difference between melting points among the methods of distinguishing amplified products from the genomic DNA sequence and the artificial DNA sequence; FIG. 7 is a view showing a kit of analyzing the copy number of a chromosome, gene or specific nucleotide sequence according to an embodiment of the invention; FIG. 8 is a view showing a kit of analyzing the copy number of a chromosome, gene or specific nucleotide sequence according to an embodiment of the invention; FIG. 9 is a graph showing a result of example 3 of the invention; FIG. 10 is a view showing a RR value determining process in example 4 of the invention; FIG. 11 is a view showing specific RR values of the example 4 of the invention; FIG. 12 is a view showing a comparison of nRR values of a normal person sample with nRR values of Down's syndrome patient, in the example 4 of the invention; FIG. 13 is a graph showing a result obtained from a MLPA method, so as to compare a result obtained from the invention with the MLPA method; and FIG. 14 is a graph showing a result obtained from a MAPH method, so as to compare a result obtained from the invention with the MAPH method. [Best Mode]

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. According to the invention, two or more genomic genes or DNA sequences are selected from sample DNA sequences and artificial SNP sequences and co- amplified, and then relative amounts of the genes or DNA sequences are determined. At this time, so as to attempt a correct quantitative analysis, the determination of relative amounts is made, based on relative amounts for the amplified products of the co-amplified artificial SNP sequences with the genes. Through the measurement having the correctness secured, it is possible to determine a change of duplication, deletion and the like of a specific chromosome in which the sample DNA exists. At this time, the genomic DNA sequences and the artificial SNP sequences are distinguished using a fact that one or more different bases exist in both the sequences. In the artificial SNP sequences, one or more bases of wild-type sequences are replaced with another base. However, remaining bases except for the replaced base are made to be same as the wild-type sequences, so that the amplification efficiency is not greatly different from that for the wild-type genomic DNA sequences during the amplification. At this time, a base change of a difference between the wild-type sequences and the artificial SNP sequences is not found or very rarely found in a normal person and made artificially. This is a means for allowing the genomic DNA sequences and the artificial SNP sequences to be distinguished after the sequences are amplified. When mixing two or more artificial SNP sequences with the sample genomic DNA sequences, it is preferred that the ratio thereof is experimentally determined to be constant in advance. The ratio is determined according to an experimental condition allowing magnitudes of signals from both sequences (wild-type sequences and artificial SNP sequences) to be similar after the amplification. As a primer is used when two or more genes are amplified with a multiplex PCR method, it is preferred to use a primer common to the wild-type and artificial SNP sequences,. In addition, during the amplification, the genes of the sample DNA and the genes of the artificial SNP sequences are competitively amplified. At this time, a relative amount of the amplified products is proportional to a relative amount of an amount of the wild-type genes and an amount of the artificial SNP sequences. By allowing two or more genes to be co-amplified, the relative amount of the two loci is calculated, based on the relative amount of the two amplified products relative to the artificial SNP sequences, so that it is possible to find out a change of the copy number of the chromosome, gene or nucleotide sequence. At this time, the amplified products made from the genomic sequences and the amplified products made from the artificial SNP sequences are distinguished by using a difference of one or more base sequences introduced artificially. This result has a relatively accurate value, compared to the other molecular methods of determining the copy number of specific genes. Specifically, one thousand or more genomic sequences and one thousand or more artificial SNP sequences are competitively amplified, and the relative amount thereof is used to measure the copy number of the chromosome, gene or nucleotide sequence, so that a change of the relative amount occuring during the amplification can be minimized. The invention can be used to diagnose and to screen the chromosome abnormality due to the duplication and deletion, such as trisomy, monosomy and sex chromosome abnormality. In addition, the invention is useful for the diagnosis on the genetic diseases due to the deletion of a small chromosome such as Duchenne muscular dystrophy and for the detection of the small chromosomal changes in diseases having inherited tendency induced by diverse causes, such as mental retardation, Alzheimer's disease and diabetes. Further, the invention can be used to analyze a change of the copy number of oncogenes and tumor suppressor genes in tumor tissues, or an abnormality of the general chromosome number. In order to describe the invention more clearly, the terms used herein are defined as follows. A "DNA" or " deoxy nucleic acid" is a deoxyribonucleotide polymer in either single or double-stranded form, including analogs of nucleotide existing naturally. A "primer" is a single stranded oligonucleotide consisting of typically 15 to 30 bases. Typically, synthesized primer is used but naturally occurring polynucleotides can also be used. A sequence of the primer is not necessarily to be the exact sequence of a template but must be sufficiently complementary to form a hybrid with the template. A primer position or primer binding site refers to a segment of the target DNA to which the primer forms the hybrid. "Hybridization" is usually performed under conditions that an oligonucleotide and a target DNA are allowed to bind specifically. "Amplification" refers to a process by which a target sequence is further synthesized. In general, the amplification repeats processes of annealing, synthesis and denaturation, wherein the synthesis process is an extension or elongation prcoess. In addition, the process is also referred to as a "polymerase chain reaction (PCR)". A multiplex" amplification refers to a process of amplifying diverse (two or more) target sequences simultaneously under same conditions. A "polymorphic site" refers to a locus at which diverse bases can be found. Usually, the SNP has at least two alleles, and a frequency thereof refers to a case of occurring 1% or more in the general public. A form of the allele occurring most frequently is referred to as a wild-type form, and a form of the allele occurring less is referred to as a mutant allele. A "single nucleotide polymorphism" (SNP) is polymorphism of a single nucleotide. A site thereof is usually preceded by and followed by highly conserved sequences. The SNP usually occurs due to substitution of one base at a specific site for another base or due to a deletion or duplication of a nucleotide. An "artificial SNP" or "aSNP" is referred to as a polymorphism of a single nucleotide, whose base change is not found or very rare in the natural world, and is artificially introduced. It is preferred that the base change naturally existing less than 1% or most preferably less than 0.1% is introduced. Since the base change naturally existing 1% or more is difficult to be distinguished from a base change occurring spontaneously, it cannot perform a function as a standard for comparing with a gene amplification amount of the genomic DNA sequence. From this point of view, it is more preferred to introduce the base change existing less than 0.1%. An "artificial SNP sequence" is referred to as a sequence including one or more artificial SNP having the base change introduced artificially. In particular, the artificial SNP sequence to be amplified referred herein is meant to the sequence same as a wild-type genomic DNA sequence, except the artificial base change. In addition to the amplified sequence with the artificial base change, the aSNP sequence might have extra sequences such as vector sequence and not amplified redundant genomic wild-type sequence. An "array" means referred that there are many target elements. Each of the target elements consists of a defined amount of biological molecules. A "sample" is a biological substance from a living organism and is mostly meant by a biological substance originating from a human. "A copy number change of chromosome, gene or specific nucleotide sequence" includes decrease as well as increase. In other reacting an extension primer capable of complementarily binding to both the artificial SNP sequence and the test sample DNA sequence, words, this includes the decrease due to the deletion of chromosome (for example, in the case of autosome, monosomy) as well as increase due to the duplication of chromosome (for example, in the case of autosome, trisomy or tetrasomy). A "wild-type genomic DNA sequence" is a representative human DNA sequence and referred to a genomic DNA sequence of which every base at all sites constituting the sequence is found in 10% or more of the normal persons. A "standard sample" is referred to as a genomic DNA sample of a normal organism without any duplication, deletion or substitution of chromosome. In addition, the genomic DNA sequence of the standard sample has the wild-type genomic DNA sequence. "aSNP normalization" means expression of the amplified gene amount of the genomic DNA sequence such as standard sample or test sample as a relative numerical value based on the amplified gene amount of the artificial SNP sequence co-amplified with the sample. A "test sample" is a subject for which the copy number of the chromosome, gene or specific nucleotide sequence is measured. Hereinafter, the invention will be more specifically described with reference to the drawings. Fig. 1 illustratively shows two artificial SNP sequences (A' and B') and two wild-type genomic DNA sequences (A and B). In A and A', only one base is different in sequences between the forward primer and the reverse primer. B and B' are the same. AF and AR are primers for amplifying A and A' gene fragments, and BF and BR are primers for amplifying B and B' genes. In Fig. 1, a base G in the wild-type genomic sequences A and B is replaced with a base T for the artificial SNP sequences A' and B'. Such one or two or more base change can be introduced as long as it does not cause a large change of efficiency during the amplification. Many methods can be used to substitute the base in the artificial SNP sequence. For example, a method of using an error occurring during the PCR amplification, a method of synthesizing oligonucleotides or a combination thereof may be used. The invention may use two or more genes to determine the gene copy number. The genes may be in a same chromosome or in different chromosomes. Fig. 2 shows a process of the invention. Fig. 2 is a schematic view showing processes of amplifying two target genes (A3 A' and B3 B') from a mixture of two kinds of sample DNA sequences and two kinds of artificial SNP sequences and analyzing a relative amount of the two target genes based on the co-amplified artificial SNP sequences. An experiment is usually progressed for a standard sample and a test sample at the same time. Alternatively, measurement for the standard sample may be firstly carried out and the measured value may be set as a standard value. After that, measurement for a test sample may be carried out whenever there are test samples and the measured values may be compared with the standard value. In Fig. 2, the standard sample and the test sample were progressed at the same time. Two or more artificial SNP sequences were mixed with the standard sample DNA (left in Fig. 2) and the test sample (right in Fig. 2), respectively, and then an experiment was simultaneously carried out in different tubes. According to the invention, it is preferred that (1) a ratio between the artificial SNP sequence and the sample DNA sequence and (2) a relative ratio of the two or more artificial SNP sequences are experimentally determined to be constant in advance. The determination is made according to an experimental condition allowing magnitudes of signals from two kinds of sequences (amplification products of wild-type DNA sequence and artificial SNP sequence) to be similar after the amplification. When a relative ratio between the artificial SNP sequences (A', B') is once determined, the mixture is mixed with the ratio and then aliquoted. The aliquoted mixture is used for the standard sample and the test sample. In addition, the relative amount between the artificial SNP sequence and the sample DNA sequence is made to be constant. As primers used to amplify the A and B genes in the mixture of the sample DNA sequence and the artificial SNP DNA sequence, AF and AR are used for the amplification of A and A' genes and BF and BR are used for the amplification of B and B' genes. According to the invention, it is possible to amplify three or more genes (A, B, C) at the same time. During the amplification, A and A' are amplified in an equal or similar efficiency and B and B' are amplified likewise, so that a relative ratio (=(A/A')/(B/B')) of A/A' and A/B' has a constant value in the normal person when the relative amount of the artificial SNP DNAs is constant. For the evaluation of the ratios of A/A or B/B', each products amplified from the genomic DNA sequences and the artificial SNP sequences need to be distinguished. At this time, it is used a base change introduced artificially. For doing so, many different methods can be employed, and two examples are shown in Figs. 4 and 5. They will be more specifically described later. Figure 3 shows an experiment result according to a method of Fig. 2. In other words, Fig. 3 shows a result obtained from the standard sample and test sample (aneuploidy sample). The relative ratio (RR) is defined as (A/A')/(B/B'). When there is a chromosomal change in the test sample (aneuploidy sample), the RR of the test sample is larger or smaller than that from the standard sample. For example, when the copy number of A gene at a starting point is larger than that of A' due to the duplication, the RR becomes different from the RR of the standard sample since the amplification amount is relatively much compared to the A' (in the case of A duplication, the RR of the test sample is larger than that of the standard sample). A normalized RR (nRR) is defined as (RR of the test sample)/(RR of the standard sample). In Fig. 3, it can be seen that since the nRR comparing the RR of the test sample with the RR of the standard sample is 2, not 1, the A gene is duplicated at first. That is, it can be seen that there is duplication in the chromosome including the A gene. When both A and B genes are in autosome and the A gene is in the trisomic chromosome, the nRR value becomes 1.5, and When there is one deletion in A gene, the nRR value becomes 0.5. In this case, it is needles to say that the B gene should be a DNA sequence of a normal chromosome having no gene changes. In order to validate that the B gene is normal, before the amplification, when it is analyzed that the ratio of the artificial SNP sequence and the test sample genomic DNA sequence, i.e., A/A or B/B' is within a certain ratio, it is possible to distinguish the duplication and deletion of both genes. When there is duplication in both A and B, both (A/A') and (B/B') values become large, and when there is deletion, both the values are smaller (but, the RR or nRR is not changed). In the above, it was analyzed for the A and B genes only. However, it is more preferable to test three or more loci at the same time. In this case, it is possible to highly increase the accuracy of the experiment result, compared to the experiment with two loci only. More preferably, loci existing in five or more different chromosomes are simultaneously tested and the values thereof can be compared. Further, it is possible to analyze many chromosomal regions at the same time through a multiplex test carrying out several tests simultaneously. The more chromosomal regions in a given chromosome are tested, the more accurate the determination for the chromosomal changes becomes. That is, when the loci to be tested are in a same chromosome, cross checks can be made. Accordingly, it is possible to judge the chromosomal changes very correctly. In addition, when the genes to be tested are in the different chromosomes, it is possible to determine rapidly and easily whether there is any genetic diseases or chromosomal changes by carrying out the tests for all chromosomes in one organism at the same time. For example, by performing many test sets designed at a time, i.e., when different 12 multiplex tests are designed, in which a multiplex unit consists of 5 loci, total 60 chromosomal regions are tested at a time. When test kits capable of measuring 500 chromosomal regions, i.e., different 100 test kits consisting of a multiplex unit of 5 are tested in a multiplex array manner using 50 wells, they can be used for karyotyping in genetic disease patients. Fig. 4 is a schematic view showing a single base extension method , one of the methods to distinguish the amplified products from the genomic DNA sequence and the artificial DNA sequence. This method can be performed with a SNaPshot kit supplied from Applied Biosystem Inc. An extension primer is capable of complementarily binding to both the artificial SNP sequence and the test sample DNA sequence, and has 3' end base to bind complementarily with the first base existing at 3' side from the base position that is artificially introduced to the artificial SNP sequence existing on a strand complementarily binding with the primer so that the first polymerization by the primer occurs at the site where the base of the genomic DNA sequence is different from that of the artificial SNP sequence. A single base is extended by hybridization of the extension primer with the amplified products using four dideoxy nucleotides differently labeled from one another and DNA polymerases (usually, Taq polymerase). The extended primer is subject to a capillary electrophoresis to measure a relative amount through the labeled signals. In the reaction, when the extension primer is extended with the four differently labeled dideoxy nucleotides and the polymerases, two products are made, which are produced by the genomic DNA sequence and the artificial SNP sequence, respectively. The two extended products are distinguished with dideoxy nucleotides labeled with differently labeled fluorescent materials. In Fig. 4, the extended products have extended G or T. For the two products, it is possible to measure the relative amount after the capillary electrophoresis (ABI 3100). Fig. 5 shows another method of distinguishing the amplified products from the genomic DNA sequence and the artificial SNP sequence. For the method, it is possible to use the extension primers as shown in Fig. 4. In Fig. 5, the extension primer s are extended with polymerase using a labeled dideoxy GTP and the unlabeled remaining three different deoxy nucleotides. At this time, two extended extension primers having different lengths are produced, each of which is formed from the genomic DNA sequence and the artificial SNP sequence. For the two products, it is possible to measure the relative amount after the capillary electrophoresis (ABI 3100). In this method, a selection about which one of the four nucleotides is used for the labeled dideoxy nucleotide is determined according to the base that is different between the artificial SNP sequence and the wild-type sequence. Any bases that are different between two sequences can be chosen for this assay. The invention includes a detection of chromosomal changes by amplifying only the signals of the sequences without amplifying the sequence itself, in addition to a detection of chromosomal changes by amplifying the sequences themselves and then comparing the amplified gene amounts. The amplification of signal can be made using a branched chain nucleotide. The signal comprises a specific binding sequence with target sequences and a tail region with several hundreds of braches suspended so that the signal can be amplified. When a specific sequence recognition portion is attached to the target DNA (genomic DNA and artificial SNP DNA) and then the labeled oligonucleotide hybridize with the braches complementarily, the relative amount of the gene copy number can be easily measured without the DNA amplification. As a method of amplifying the signal, any suitable methods known and any method capable of amplifying the signal can be used. Fig. 6 is a schematic view showing a method of using a difference between melting points (melting curve analysis) that is one of the methods of distinguishing amplified products from the genomic DNA sequence and the artificial DNA sequence (melting curve analysis). This method uses the fact that the melting points of the hybrid between the amplified sequences and the probes are changed by a base change introduced artificially. A probe is designed to be complementary to the sequences amplified from the genomic sequences and to produce a signal when it binds to a complementary sequence. In addition, the probe is designed to bind complementarily with the aSNP sequences but to have a lower melting point as the complementary bond is broken at a changed site introduced artificially. It is designed that a measurable signal can be detected even when the probe binds with a product from the artificial SNP sequence. When the signal is measured from the hybrid formation between the amplified product and the probe while increasing or decreasing the temperature of the reactant gradually, a relative amount of the two different products can be evaluated. On the other hand, the probe can be designed to bind complementarily to the genomic DNA sequence and now have lower melting point when the binding with the artificial SNP sequence. When the signal is measured from the hybrid formation between the amplified product and the probe while increasing or decreasing the temperature of the reactant gradually, a relative amount of the two different products can be evaluated. In analyzing the melting point, it is possible to individually analyze the co-amplified genes or to simultaneously carry out the melting point analysis for two or more genes using different fluorophores. In the melting point analysis method, the RR and nRR values are also used for determining the chromosome aberration, likewise the above methods. In Figs. 4 to 6, any fluorophores can be used as the labeled compounds, such as FAM, ROX, TAMRA, RIlO, R6G, Joe, HEX, TIETI, Alexa, Cy3 and Cy6 (Gene Link, www, genelink.com; AnaSpec inc., www.anaspec.com; Eurogentec, www.eurogentec.com; Synthegen LLC, www.synthegen.com") Hereinafter, preferred embodiments of the invention will be more specifically described. It should be noted that the invention is not limited to the following embodiments but to the claims.

[Mode for Invention]

<Example 1: a kit for measuring the copy number of chromosome, gene or specific nucleotide sequence> Fig. 7 is an example of a kit for measuring the copy number of chromosome, gene or specific nucleotide sequence, particularly an example of a kit for measuring the copy number of all chromosomes in a single organism. In this example, a well of a plate 1 contains primers capable of multiplex amplification of the loci and the artificial SNP sequences. Each of the wells is designed in order for a combination of different loci to be co-amplified. For example, when each well is designed to amplify five different loci, 480 kinds (5 kinds x 96 wells) of loci can be simultaneously analyzed. A predetermined amount of sample DNA obtained from a person or tissue is aliquoted in a same amount in each of the wells and a reagent A for amplifying the gene is also aliquoted in each well. After the amplification, the amplified product is validated with the electrophoresis and the remaining primers are removed. A portion of the amplified product is put into another plate 2 and subject to the single base extension method. Each well of the plate 2 contains 96 combinations of extension primers so that the single base can be extended in the amplified product. An enzyme for the single base extension, a labeled ddNTP, a buffered solution and the like may be comprised of independent reagents. When the single base extension reaction is finished, the capillary electrophoresis is carried out to measure a kind and a relative amount of the extended single bases. The plate 2 may consist of other means capable of measuring the relative amount of the amplified products from the artificial SNP and genomic DNA, in addition to means for the single base extension. An analysis of amplification or deletion of a specific loci from the raw data analyzed by the electrophoresis can be made with a series of automated software, and it is possible to analyze where the chromosomal regions the amplification or deletion occurs. In the process of the analysis, the standard samples are analyzed in advance to determine ratios of the genes to be relatively amplified from the genomic DNA and the artificial SNP sequences at every locus and data having the cutoff values determined in advance is used, so that the deletion and amplification of the specific loci can be analyzed.

<Example 2: a kit for measuring the copy number of chromosome, gene or specific nucleotide sequence> Fig. 8 is an example of a kit for measuring the copy number of chromosome, gene or specific nucleotide sequence, according to an example of the invention. In this example, although the single base extension method is shown to distinguish the gene products amplified from the aSNP and the genomic DNA, other method can be used. In the reagent A, mixtures of the artificial SNP sequences and mixtures of the primers for the multiplex amplification are mixed. Reagent B contains a mixture of the enzyme used for the gene amplification and deoxynucleotide triphosphaste and a buffered solution, and each component of the reagent B may be comprised of individual reagents. The reagents A and B are mixed in a certain amount and aliquoted in each of the tube. At this time, in addition to the experiment for the test sample, a tube for the standard sample and a tube for chromosome aberration sample may be prepared and subject to an experiment together. The separated genomic DNA originated from the test chromosome for which the chromosome copy number is measured is put into the tube except the first and second tubes, and the standard sample DNA and the chromosome aberration DNA are added to the first and second tubes, respectively. After the gene amplification, it is validated using the electrophoresis whether the gene is amplified. And the remaining primers for the amplification are removed. All of the processes can be automated in the kit. A portion of the amplified product is mixed with the primers for the single base extension, and the single base extension is carried out. This procedure is one for measuring a relative amount of the amplified products from the artificial SNP or genomic DNA. The other methods may be used instead of the single base extension. The single base extended primers are subjected to capillary electrophoresis, and the extended bases are confirmed and the relative amount is analyzed. <Example 3: measurement of the gene copy number using DSCRl and hexokinase genes> A DSCRl gene is located at the 21th chromosome of the human and a hexokinase gene is located at the 10th chromosome of the human. Segments of the DSCRl gene were amplified with the PCR method using the following primers: DF (GCC AAA TCC AGA CAA GCA GTT TC) and DR (GAT CAG CCG CAG TCT CTC TAA CAC). During the amplification, in order to induce an occurrence of error, the PCR amplification with 40 cycles was repeated 3-5 times according to the following conditions, while diluting every time: 940C for 30s, 550C for 30s and 72°C for 30s. The artificial SNP sequence contained the T instead of G that is the 316th base of the amplified PCR product. The hexokinase gene was amplified with the PCR method using the following two primers: HF (TCT GGG CTC TTG TCC AGT ATT GAG T) and HR (ATT CCA ACC CTC CCT CCT GAG T). During the amplification, in order to induce an occurrence of error, the PCR amplification with 40 cycles was repeated 3-5 times under the same conditions as the DSCRl amplification, while diluting every time. The artificial SNP sequence contained the T instead of G that is the 211th base of the amplified PCR product. All of the artificial SNP sequences were cloned to a T-vector (pCRII, Invitrogen). The genomic DNA was separated from bloods of the wild-type and the test samples, respectively. Each of the purified DNA (50 ng) was mixed with 300 fg of the artificial SNP hexokinase-pCRll and 480 fg of the artificial SNP DSCRl-pCRΪl and then left at 980C for 5 minutes. The mixture of the sample DNA and the two artificial SNP DNAs was subject to the multiplex PCR with 35 cycles using the four kinds of primers, i.e., DF, DR, HF and HR, according to the following conditions: 950C for 30s, 59°C for 30s and 720C for 30s. The amplified PCR product was purified using a GeneClean kit (Bio 101), and then the single base extension was carried out using SNaPshot kit (Applied Biosystems) wherein the following primers were used: DE (GCC TCT TGG CAC CAC CT) for DSCRl and HE (GTT GTA AGC CCT CAG CAG TT) for hexokinase. 25 cycles were used for the single base extension reaction: 96°C for 10s, 5O0C for 5s and 6O0C for 30s. The single base extended products were analyzed using ABI 3100 (Applied Biosystems). As shown in the graph of Fig. 9, in six wild-type samples obtained from different sources, respectively, it was calculated an average (RRaverage, i.e., Rl/R2average) of RR (R1/R2) values of final products from the DSCRl and hexokinase. RRaverage was 0.865 and a standard deviation was 0.016. To the contrary, RR (R1/R2) of the test genomic DNA, i.e., RRpatient was 1.28 and normalized RR value, i.e., nRR value (=RRpatient/RRaverage) was about 1.5 (1.28/0.865). Accordingly, the 21th chromosome was determined to be trisomy having 1.5 times duplication.

<Example 4: measurement of the gene copy number using DSCRl, hexokinase, RbI and DCC genes> A DSCRl gene is located on the 21th chromosome, a hexokinase gene is located on the 10th chromosome, a RbI gene is located on the 13th chromosome and a DCC gene is located on the 18th chromosome. The artificial SNP sequences of the DSCRl and hexokinase genes were same as those used in the example 3. The RbI and DCC genes were respectively amplified with RbF (AGG ACC CTA ACA CAG TAT ATC CCA AGT G), RbR (GAA ATA ATG TGG CTT TGA ACA TGC CAG T), DCCF (CCT GGC TGT GGT CTC CTA GGT CAG ACT T) and DCCR (GAG TCC TTC CAA ACT TGC CAT TTG TTC A) and then cloned to the T-vector (pCRII, Invitrogen). It was found a clone of which the C, which is the 44th base of the RbI amplified, was replaced with the A and a clone of which the G, which is the 138th base of the DCC amplified, was replaced with the T and then the clones were used as each aSNP sequence. The genomic DNA was separated from bloods of the normal person and the Down's syndrome patient. Each of the purified DNA (20 ng) was mixed with 300 fg of the artificial SNPs of hexokinase, DSCRl, RbI and DCC, respectively and then left at 98°C for 5 minutes. The mixture of the sample DNA and the four artificial SNP DNAs was subject to the multiplex PCR with 32 cycles using the eight kinds of primers for amplifying the four genes, i.e., DF, DR, HF, HR, RbIF, RbIR, DCCF and DCCR according to the following conditions: 95°C for 30s, 590C for 30s and 72°C for 30s. The amplified PCR product was purified using a PCR purification kit (Qiagen), and then the single base extension was carried out using SNaPshot kit (Applied Biosystems) wherein the following extension primers were used: DE (GCC TCT TGG CAC CAC CT) for DSCRl, HE (GTT GTA AGC CCT CAG CAG TT) for hexokinase, RbE (TCA TTT GTA TCT TAA TTC TTC AGG ACC C) for RbI and DCCE (CTC TCA TTC TCT TTA TAG GTG TAT GAA CA) for DCC. Fiften cycles were used for the single base extension with the following condition: 960C for 10s, 500C for 10s and 600C for 30s. The single base extended products were analyzed using ABI 3100 (Applied Biosystems). In the standard samples of 20 normal persons, the peak ratios (in Fig. 10, Rl, R2, R3 and R4) of signals, which were obtained from the amplified product of DSCRl, hexokinase, RbI and DCC each of which origin is either from genomic DNA or aSNP sequences. And each of the RRpatients (relative ratio: R1/R2, R1/R3, R1/R4, R2/R3, R2/R4 and R3/R4) and then RRaverage (Rl/R2average, Rl /R3 average...), an average value of each standard sample RR values were obtained. The nRR (normalized RR = RRpatient/RRaverage) of each of Down's syndrome patients was obtained using the RRaverages. The result is shown in Fig. 12. The standard deviation (SD) of the RRaverage was 0.029-0.043. All nRRs of the 20 normal persons were 0.9-1.1 and the nRRs of the 10 Down's syndrome patients were as follows: nRlR2 (normalized R1/R2), nRlR3 (normalized R1/R3) and nRlR4 (normalized R1/R4) were 1.4-1.6 and all of the remaining nRRs were 0.9-1.1. From the above result, in the case of Down's syndrome patients, there was 1.5 times increase of the chromosome copy number in the 21th chromosome on which the DSCRl gene is located. Likewise, it could be re-validated that the Down's syndrome patient had a trisomy chromosome aberration. As shown in the example 4, the invention exhibited the remarkably high accuracy, compared to the other molecular methods such as MLPA (Multiplex Ligation dependent Probe Amplification) or MAPH (Multiplex Amplifiable Probe Hybridization). In the graph of Fig. 12, it can be seen that all nRR values of the 20 normal person samples are not deviated from a range of about 0.9 — - 1-1- In addition, the nRR value of the normal gene among nRR values of the Down's syndrome sample was within a range of 0.9 to 1.1 (1+0.1), and all of the nRR values of the trisomy gene were within a range of 1.4 to 1.6 (1.5+0.1), except one patient. Fig. 13 is a graph showing a quantitative MLPA analysis of the normal samples. A box indicates an analysis value range of 50% of the whole samples, and a straight line indicates an analysis value range of 95% of the whole samples. The graph shows distributions of personal probes specific at 13th, 18th, and 21th chromosomes (n=474). The standard deviation is 0.13-0.3 according to the probes. Contrary to the example 4 of the invention, according to the MLPA method, the analysis value of the normal sample is 0.5-1.7, as shown in Fig. 13. Further, the box shows an area to which 50% sample belongs and is 0.78 to 1.23. This is remarkably contrasted with the analysis value of the normal person, i.e., 0.9-1.1 of this invention. Fig. 14 is a graph quantitatively analyzing signal intensities obtained from two separate experiments which examined twelve DNA samples (ten organisms), total 40 probes using the MAPH method. The autosome probe and feminine X- coupling probe were distributed at about 1.0, and the male X-coupling probe was concentrated at the surrounding of 0.5. As shown in Fig. 14, it can be seen that the MAPH method has a remarkably poor correctness compared to the invention. In the analysis value of the normal sample, 'a' ranges from 0.65 to 1.45, 'b' ranges from 0.7 to 1.3. In the analysis value of the test sample having the deletion, 'b' ranges from 0.55 to 0.75, not including 0.5. Further, the analysis value of the normal sample appears even at the middle (0.65) of the analysis value range of the test sample having the deletion. This means that the MAPH has a remarkably poor accuracy compared to the invention.

[Industrial Applicability] As described above, according to the invention, it is possible to obtain more accurate value than the other molecular methods determining the copy number of the specific genes as well as to remarkably reduce the time and necessary manpower, when analyzing the change of the chromosome.