MICROARRAY CHIP AND METHOD FOR DETECTION OF CHROMOSOMAL ABNORMALITY

Information

  • Patent Application
  • 20100210469
  • Publication Number
    20100210469
  • Date Filed
    September 07, 2007
    17 years ago
  • Date Published
    August 19, 2010
    14 years ago
Abstract
The present invention relates to techniques to detect chromosomal columns abnormalities. More specifically, the present invention is directed to a microarray chip for detecting chromosomal abnormalities comprising one or more pooled probe sets, wherein the pooled probe set is specific to a chromosomal abnormality and all probes of each pooled probe set are immobilized together in at least one spot; a method of detecting chromosomal abnormalities using the microarray chip; a kit for diagnosing diseases associated with chromosomal abnormalities comprising the microarray chip; and a method of diagnosing a disease associated with a chromosomal abnormality by identifying the chromosomal abnormality specific to the disease using the microarray chip. The present invention relates to techniques to detect chromosomal abnormalities. More specifically, the present invention is directed to a microarray chip for detecting chromosomal abnormalities comprising one or more pooled probe sets, wherein the pooled probe set is specific to a chromosomal abnormality and all probes of each pooled probe set are immobilized together in at least one spot; a method of detecting chromosomal abnormalities using the microarray chip; a kit for diagnosing diseases associated with chromosomal abnormalities comprising the microarray chip; and a method of diagnosing a disease associated with a chromosomal abnormality by identifying the chromosomal abnormality specific to the disease using the microarray chip.
Description
BACKGROUND OF THE INVENTION

(a) Field of the Invention


The present invention relates to techniques to detect chromosomal abnormalities. More specifically, the present invention is directed to a microarray chip for detecting chromosomal abnormalities comprising one or more pooled probe sets, wherein the pooled probe set is specific to a chromosomal abnormality and all probes of each pooled probe set are immobilized together in at least one spot; a method of detecting chromosomal abnormalities using the microarray chip; a kit for diagnosing diseases associated with chromosomal abnormalities comprising the microarray chip; and a method of diagnosing a disease associated with a chromosomal abnormality by identifying the chromosomal abnormality specific to the disease using the microarray chip.


(b) Description of the Related Art


Chromosomal abnormality is associated with genetic defect and degenerative disease. The chromosomal abnormality can be a deletion or duplication of a chromosome, a deletion or duplication of a part of chromosome, or a break, translocation, or inversion in the chromosome. The chromosomal abnormality is a disturbance in the genetic balance and causes fetal death or serious defect in physical and mental states. For examples, Down's syndrome is a common abnormality of chromosome number caused by the presence of a third chromosome 21 (trisomy 21). Edwards syndrome (trisomy 18), Patau syndrome (trisomy 13), Turner syndrome (XO) and Klinefelter syndrome (XXY) also belong to abnormalities in chromosome number.


The chromosomal abnormality can be detected by using Karyotype, and Fluorescent In Situ Hybridization (FISH). These detection methods have disadvantages in terms of time, labor and accuracy. Moreover, the Karyotype requires much time in cell culture. FISH can only be used for samples where the nucleic acid sequence and chromosomal location are known. Comparative genome hybridization (CGH) can be used to avoid problems of FISH. CGH can analyze a whole genome to detect the part where chromosome number abnormality occurs. However, the disadvantage of CGH has a low resolution compared to FISH.


In a different approach, DNA microarrays can be used for detecting the chromosomal abnormality. DNA microarray systems may be classified into cDNA microarrays, oligonucleotide microarrays, and genome microarrays, depending upon the kinds of the bio-molecules immobilized on the microarray. Even though cDNA microarrays and oligonucleotide microarrays are easily prepared, the systems have the disadvantages of the limitation in the number of probes immobilized on the microarray, high cost of probe preparation, and difficulty in detecting a chromosomal abnormality located external to the probe. In particular, for genomic DNA microarray systems, although the probe can be easily made, and can detect chromosomal abnormalities in the expansive area of the chromosome, and also in intron areas of the chromosome, it is difficult to prepare a large number of DNA fragments where the chromosomal location and function are identified.


Therefore, it has been required to develop techniques to easily detect chromosomal abnormalities with high performance.


SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a microarray chip for detecting chromosomal abnormality comprising one or more pooled probe sets, wherein each pooled probe set is specific to a chromosomal abnormality and immobilized in one spot.


In another embodiment, the present invention provides a method of preparing the microarray chip comprising the step of immobilizing one or more pooled probe sets in spots on a substrate, wherein each pooled probe set is specific to a chromosomal abnormality, and immobilized in one spot.


In yet another embodiment, the present invention provides a method of detecting chromosomal abnormalities using the microarray chip.


In yet another embodiment, the present invention provides a kit for diagnosing a disease associated with a chromosomal abnormality, comprising the microarray chip.


In yet another embodiment, the present invention provides a method of diagnosing a disease associated with a chromosomal abnormality using the microarray chip.


The disease diagnosable by the present invention may be one or more selected from the group consisting of Down syndrome, Patau syndrome, Edward syndrome, Tuner syndrome, Klinefelter syndrome, Super female syndrome, Super male syndrome, Wolf-Hirschhorn syndrome, Cri-Du-Chat syndrome, William syndrome, Prader-willi syndrome, Angelman syndrome, Miller-Dieker Lissencephaly syndrome, Smith-Magenis syndrome, Digeorge syndrome, Steroid Sulfatase deficiency, and the like.





BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention, and many of the attendant advantages thereof, will be readily apparent as the same becomes better understood by reference to the following detailed description when considered in conjunction with the accompanying drawing, wherein:



FIG. 1 is a schematic view illustrating the present microarray according to an embodiment.



FIG. 2 exemplarily shows pooling of BAC clones to generate pooled Chromosome 1 probe set.



FIG. 3 shows the regions (orange color) used as controls in Example 2.5.



FIG. 4 shows the FISH result using Chromosome 13 as target and #626(13q12.1) as control in Example 2.5.



FIG. 5 shows the FISH result using Chromosome 13 as target and Cen. 7 as control in Example 2.5.



FIG. 6 shows the FISH result using Chromosome 21 as target and #88(21q11.2) as control in Example 2.5.



FIG. 7 shows the FISH result using Chromosome 21 as target and Cen. 7 as control in Example 2.5.



FIG. 8 shows the FISH result using Chromosome X as target and Cen. X as control in Example 2.5.



FIG. 9 shows the results of λHindIII Marker Separation in 1% agarose gel, wherein the numbers indicated at right of the column correspond to those of Table 39.



FIG. 10 schematically shows the detection processes of the present invention.



FIGS. 11A and 11B show the results obtained in Example 3.





DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A more complete appreciation of the invention, and many of the attendant advantages thereof, will be readily apparent as the same becomes better understood by reference to the following detailed description.


In one aspect, the present invention relates to a microarray chip for detecting chromosomal abnormality comprising one or more pooled probe sets, wherein each pooled probe set is specific to a chromosomal abnormality and immobilized in one or more spots.


In the present invention, the chromosomal abnormality may include aberrations in copy number of chromosome associated with aneuploidy of one or more Chromosomes 1 to 22, X and Y, or quantitative aberrations caused by micro-deletion or micro-duplication of a specific chromosomal region. Generally, such chromosomal abnormality in chromosomal copy number and/or a specific chromosomal region is associated with various diseases. The representative examples of such chromosomal abnormality-associated diseases are summarized in following Table 1.











TABLE 1






Chromosomal abnormality



Disease
(Karyotype)
Incidence







Down Syndrome
2n = 47 (trisomy 21)
1/770


Patau Syndrome
2n = 47 (trisomy 13)
1/15,000


Edward Syndrome
2n = 47 (trisomy 18)
1/4,000~1/8,000


Tuner Syndrome
2n = 45 (XO)
1/10,000 females


Klinefelter Syndrome
2n = 47 (XXY)
1/1000 males


Super female syndrome
2n = 47 (XXX)
1/1000 females


Super male syndrome
2n = 47 (XYY)
1/1000 males


Wolf-Hirschhorn syndrome (WHS)
4p16.3 deletion
1/30,000~1/50,000


Cri-Du-Chat syndrome
5p15.2 deletion
1/30,000~1/50,000


William syndrome (WBS)
7q11.2 deletion
1/7,500~1/20,000


Prader-willi syndrome (PWS)
15q11-15q13 deletion (paternal)
1/10,000~1/15,000


Angelman syndrome (AS)
15q11-15q13 deletion(maternal)
1/15,000~1/30,000


Miller-Dieker Lissencephaly syndrome
17p13.3 deletion
1/100,000


(MDLS)


Smith-Magenis syndrome (SMS)
17p11.2 deletion
1/25,000


Digeorge syndrome (DGS)
22q11.2 deletion
1/4,000~1/6,000


Steroid Sulfatase deficiency syndrome
Xp22.31 deletion
1/2,000~1/6,000 males


(STS)









As used herein, the term ‘probe’ refers to a nucleic acid fragment which is immobilized on a microarray and capable of hybridizing with a homologous DNA in a test sample or reference. The probe can be DNA, RNA, cDNA or mRNA, or oligomer DNA. Preferably, the probe has a single chromosomal locus.


In an embodiment of the present invention, the probes may be selected from Bacterial Artificial Chromosome (BAC) clones. The BAC clones include BAC vectors containing a certain size fragment of the whole human genome. Because a BAC clone includes one fragment of a human DNA, the BAC clone corresponding to a specific part of a chromosome can be arrayed by analyzing the nucleic acid sequence and chromosomal location of the inserted DNA. Thus, specific DNA fragments can be easily obtained from each BAC clone.


As used herein, the term ‘pooled probe set’ refers to a mixture of probes, preferably, in equal amounts, wherein the probes are specifically selected from the whole genome DNA, preferably human genome, as representing each chromosome or a specific chromosomal region and being capable of specifically detecting the genetic status thereof, such as, the copy number change, micro-deletion, micro-amplification, and the like. In the present invention, 32 pooled probe sets are identified for Chromosomes 1 to 22, X and Y, and several chromosomal regions associated with micro-deletion, respectively.


The most important feature of the present invention resides in that several probes representing a chromosome or a chromosomal region are specifically selected and pooled for each chromosome or chromosomal region, for the use in detecting a specific chromosomal abnormality, to achieve a rapid, convenient and accurate detection.


As described above, the pooled probe sets of the present invention are specifically selected for a specific type of chromosomal abnormality, and thus, specifically related to the chromosomal abnormality-associated disease.


The pooled probe set may be one or more selected form the group consisting of:


a pooled probe set (pooled probe set 1) specific to the chromosomal abnormality in copy number of Chromosome 1 consisting essentially of human chromosomal polynucleotides carried in BAC27_N16, BAC25_C19, BAC153_I07, BAC217_C19, BAC59_D13, BAC54_I02, BAC163_C09, BAC218_G03, BAC152_F22, BAC34P03, BAC36_I16, BAC145_L11, BAC37_O23, BAC239_G19, BAC105_P13, BAC57_N17, BAC239_A12, BAC171_H09, and BAC222 E02;


a pooled probe set (pooled probe set 2) specific to the chromosomal abnormality in copy number of Chromosome 2 consisting essentially of human chromosomal polynucleotides carried in BAC126_E04, BAC197_E10, BAC43_A02, BAC33_C05, BAC59_D21, BAC12_G01, BAC141_F07, BAC163_C22, BAC36_H22, BAC143_G24, BAC238_G01, BAC252_A16, BAC46_J12, BAC57_C12, BAC34_F17, BAC79_L21, BAC39_M07, BAC156_K09, BAC195106, and BAC88_K20;


a pooled probe set (pooled probe set 3) specific to the chromosomal abnormality in copy number of Chromosome 3 consisting essentially of human chromosomal polynucleotides carried in BAC197_B21, BAC158_C03, BAC144_C11, BAC186_N05, BAC103_F06, BAC114_B23, BAC102_E23, BAC119_G21, BAC68_H20, BAC237_M11, BAC168_G04, BAC61_M02, and BAC36_N19;


a pooled probe set (pooled probe set 4) specific to the chromosomal abnormality in copy number of Chromosome 4 consisting essentially of human chromosomal polynucleotides carried in BAC60_H08, BAC26_C10, BAC68_O19, BAC102_G08, BAC127_B16, BAC176_G14, BAC41_O05, BAC37_H04, BAC115_C13, BAC30_N21, BAC220_D24, BAC106_P17, BAC41_O11, BAC157_P10, and BAC27_L15;


a pooled probe set (pooled probe set 5) specific to the chromosomal abnormality in copy number of Chromosome 5 consisting essentially of human chromosomal polynucleotides carried in BAC86_B20, BAC33_N18, BAC55_L24, BAC226_H03, BAC156_E24, BAC237_B02, BAC29_D17, BAC139_M23, BAC21_J16, BAC27_N23, BAC148_D23, BAC186L21, BAC238_E21, and BAC175_N07;


a pooled probe set (pooled probe set 6) specific to the chromosomal abnormality in copy number of Chromosome 6 consisting essentially of human chromosomal polynucleotides carried in BAC125_G09, BAC182_E20, BAC81_C08, BAC24_P12, BAC76_A23, BAC26_F16, BAC43_M14, BAC27_P17, BAC1_N23, BAC247_D17, BAC101_M04, BAC90_F08, BAC118_M18, and BAC179_N12;


a pooled probe set (pooled probe set 7) specific to the chromosomal abnormality in copy number of Chromosome 7 consisting essentially of human chromosomal polynucleotides carried in BAC231_L03, BAC82_L17, BAC218_N01, BAC5_A09, BAC170_M16, BAC119_K16, BAC248_P06, BAC96_F02, BAC139_J04, BAC76_K13, BAC192_N04, BAC154_A21, and BAC120_I09;


a pooled probe set (pooled probe set 8) specific to the chromosomal abnormality in copy number of Chromosome 8 consisting essentially of human chromosomal polynucleotides carried in BAC150_M15, BAC149_J08, BAC63_M21, BAC147_O15, BAC44_I16, BAC30_N24, BAC43_J01, BAC234_M17, BAC68_K11, BAC200_C08, BAC237_M08, BAC61_N10, BAC80_H19, BAC150_P12, and BAC66_I02;


a pooled probe set (pooled probe set 9) specific to the chromosomal abnormality in copy number of Chromosome 9 consisting essentially of human chromosomal polynucleotides carried in BAC80_F23, BAC28_L14, BAC137_L16, BAC161_C10, BAC92_D01, BAC163_H11, BAC12_E22, BAC172_D10, BAC149_L08, BAC188_O18, and BAC126_N07;


a pooled probe set (pooled probe set 10) specific to the chromosomal abnormality in copy number of Chromosome 10 consisting essentially of human chromosomal polynucleotides carried in BAC 170_F05, BAC102_J19, BAC40_P04, BAC141_E23, BAC246_I22, BAC14_K16, BAC52_B14, BAC158_C10, BAC155_O18, BAC144_E19, BAC218_E11, BAC48_I12, and BAC182_N07;


a pooled probe set (pooled probe set 11) specific to the chromosomal abnormality in copy number of Chromosome 11 consisting essentially of human chromosomal polynucleotides carried in BAC68_K10, BAC90_E18, BAC24_K17, BAC58_O19, BAC36_K05, BAC150_P20, BAC154_H22, BAC26_C09, BAC119_O13, BAC195_O14, BAC73_E17, BAC142_K09, and BAC65_D19;


a pooled probe set (pooled probe set 12) specific to the chromosomal abnormality in copy number of Chromosome 12 consisting essentially of human chromosomal polynucleotides carried in BAC60_I23, BAC121_P21, BAC199_G02, BAC65_G10, BAC41_I18, BAC10_M07, BAC39_O14, BAC144_K11, BAC178_M15, BAC134_M17, BAC65_I21, and BAC27_E08;


a pooled probe set specific (pooled probe set 13) to the chromosomal abnormality in copy number of Chromosome 13 consisting essentially of human chromosomal polynucleotides carried in BAC28_H21, BAC163_F01, BAC78_C21, BAC135_O03, BAC237_P24, BAC84_N09, BAC8_C18, BAC133_G23, and BAC116_B15;


a pooled probe set (pooled probe set 14) specific to the chromosomal abnormality in copy number of Chromosome 14 consisting essentially of human chromosomal polynucleotides carried in BAC236_F24, BAC22_E01, BAC37_K09, BAC79_J20, BAC50_I09, BAC15_E12, BAC63_O11, BAC11_N10, BAC39_P02, and BAC101_O15;


a pooled probe set (pooled probe set 15) specific to the chromosomal abnormality in copy number of Chromosome 15 consisting essentially of human chromosomal polynucleotides carried in BAC66_K21, BAC162_K11, BAC178_K16, BAC21_K13, BAC167_M02, BAC88_F18, BAC168_F12, BAC10_E08, BAC177_H09, and BAC41_K03;


a pooled probe set (pooled probe set 16) specific to the chromosomal abnormality in copy number of Chromosome 16 consisting essentially of BAC38_I04, BAC96_J19, BAC120_K24, BAC177_P23, BAC247_B03, BAC117_H14, BAC96_G02, BAC24_D17, and BAC223_D19;


a pooled probe set (pooled probe set 17) specific to the chromosomal abnormality in copy number of Chromosome 17 consisting essentially of human chromosomal polynucleotides carried in BAC200_M05, BAC50_A03, BAC149_H11, BAC29_G13, BAC238_E06, BAC150_O15, BAC70_P11, BAC70_N11, BAC116_E10, and BAC48_K14;


a pooled probe set (pooled probe set 18) specific to the chromosomal abnormality in copy number of Chromosome 18 consisting essentially of human chromosomal polynucleotides carried in BAC57_H08, BAC141_I04, BAC252_H16, BAC232_E19, BAC149_I18, BAC186_P19, BAC151_L02, BAC230_C11, BAC43_A24, and BAC184_J04;


a pooled probe set (pooled probe set 19) specific to the chromosomal abnormality in copy number of Chromosome 19 consisting essentially of human chromosomal polynucleotides carried in BAC178_L22, BAC160_C11, BAC131_N13, BAC54_N22, BAC233_K14, BAC162_K04, BAC76_E22, BAC211_B15, BAC101_H02, and BAC193_C07;


a pooled probe set (pooled probe set 20) specific to the chromosomal abnormality in copy number of Chromosome 20 consisting essentially of human chromosomal polynucleotides carried in BAC247_K09, BAC26_J24, BAC75_H16, BAC37_M13, BAC19_G17, BAC82_B07, BAC96_H08, BAC166_J02, BAC41_E11, and BAC146_N07;


a pooled probe set (pooled probe set 21) specific to the chromosomal abnormality in copy number of Chromosome 21 consisting essentially of human chromosomal polynucleotides carried in BAC102_F10, BAC240_M07, BAC200_O02, BAC97_O19, BAC119_K07, BAC200_A23, BAC221_D22, BAC100_D11, BAC33_D15, and BAC126_M10;


a pooled probe set (pooled probe set 22) specific to the chromosomal abnormality in copy number of Chromosome 22 consisting essentially of human chromosomal polynucleotides carried in BAC169_G07, BAC153_I19, BAC100_P10, BAC37_J03, BAC187_K08, BAC131_H09, BAC106_C07, BAC66_M06, BAC51_M21, and BAC153_O04;


a pooled probe set (pooled probe set 23) specific to the chromosomal abnormality in copy number of Chromosome X consisting essentially of human chromosomal polynucleotides carried in BAC70_N16, BAC22_H14, BAC65_L14, BAC151_A03, BAC49_G05, BAC130_K20, BAC103_N15, BAC136_M01, BAC6_B17, BAC141_P03, BAC246_K02, BAC91_J24, BAC97_C11, BAC63_G23, BAC73_B07, BAC162_B10, and BAC119_C15;


a pooled probe set (pooled probe set 24) specific to the chromosomal abnormality in copy number of Chromosome Y consisting essentially of human chromosomal polynucleotides carried in BAC24_K23, BAC205_L13, BAC127_H21, BAC192_M14, BAC101_I21, BAC140_H17, BAC65_J16, BAC180_K16, BAC102_F03, BAC31_L01, and BAC240_H05;


a pooled probe set (pooled probe set 25) specific to micro-deletion of 4p16.3 of Chromosome 4 consisting essentially of human chromosomal polynucleotides carried in BAC50_H08, BAC67_I12, BAC100_E03, BAC1_F06, BAC135_O20, and BAC153_J14;


a pooled probe set (pooled probe set 26) specific to micro-deletion of 5p15.2 of Chromosome 5 consisting essentially of human chromosomal polynucleotides carried in BAC143_N22, BAC206_I13, BAC252_N08, BAC64_P22, BAC208_N21, BAC200_E05, and BAC240_K06;


a pooled probe set (pooled probe set 27) specific to micro-deletion of 7q11.2 of Chromosome 7 consisting essentially of human chromosomal polynucleotides carried in BAC69_O08, BAC66_N22, BAC180_N24, BAC67_C05, BAC183_A12, and BAC123_D05;


a pooled probe set (pooled probe set 28) specific to micro-deletion of 15q11-15q13 of Chromosome 15 consisting essentially of human chromosomal polynucleotides carried in BAC188_N24, BAC223_H02, BAC217_F02, BAC71_A18, BAC5_L18, BAC248_C13, BAC78_F07, BAC180_J22, BAC21_O06, and BAC105_L07;


a pooled probe set (pooled probe set 29) specific to micro-deletion of 17p13.3 of Chromosome 17 consisting essentially of human chromosomal polynucleotides carried in BAC95_J10, BAC75_C17, BAC110_O13, BAC63_J08, BAC190_F10, BAC186_M15, BAC183_M06, BAC135_N07, BAC_F06, and BAC31_H03;


a pooled probe set (pooled probe set 30) specific to micro-deletion of 17p11.2 of Chromosome 17 consisting essentially of human chromosomal polynucleotides carried in BAC249_G12, BAC41_D18, and BAC186_E14;


a pooled probe set (pooled probe set 31) specific to micro-deletion of 22q11.2 of Chromosome 22 consisting essentially of human chromosomal polynucleotides carried in BAC124_E21, BAC196_A22, BAC69_P21, BAC141_K20, BAC169_K21, BAC145_P12, and BAC224_F10; and


a pooled probe set (pooled probe set 32) specific to micro-deletion of Xp22.31 of Chromosome X consisting essentially of human chromosomal polynucleotides carried in BAC221_A12, BAC191_E24, and BAC231_F19.


In an embodiment, the present invention relates to a microarray chip comprising one or more pooled probe sets selected from pooled probe sets 1 to 24 to detect any chromosomal abnormality in copy number of Chromosomes 1 to 22, X and Y corresponding to the used pooled probe set.


More specifically, the present invention may relate to a microarray chip comprising pooled probe set 1 to detect chromosomal abnormality in copy number of Chromosome 1. The present invention may relate to a microarray chip comprising pooled probe set 2 to detect chromosomal abnormality in copy number of Chromosome 2. The present invention may relate to a microarray chip comprising pooled probe set 3 to detect chromosomal abnormality in copy number of Chromosome 3. The present invention may relate to a microarray chip comprising pooled probe set 4 to detect chromosomal abnormality in copy number of Chromosome 4. The present invention may relate to a microarray chip comprising pooled probe set 5 to detect chromosomal abnormality in copy number of Chromosome 5. The present invention may relate to a microarray chip comprising pooled probe set 6 to detect chromosomal abnormality in copy number of Chromosome 6. The present invention may relate to a microarray chip comprising pooled probe set 7 to detect chromosomal abnormality in copy number of Chromosome 7. The present invention may relate to a microarray chip comprising pooled probe set 8 to detect chromosomal abnormality in copy number of Chromosome 8. The present invention may relate to a microarray chip comprising pooled probe set 9 to detect chromosomal abnormality in copy number of Chromosome 9. The present invention may relate to a microarray chip comprising pooled probe set 10 to detect chromosomal abnormality in copy number of Chromosome 10. The present invention may relate to a microarray chip comprising pooled probe set 11 to detect chromosomal abnormality in copy number of Chromosome 11. The present invention may relate to a microarray chip comprising pooled probe set 12 to detect chromosomal abnormality in copy number of Chromosome 12. The present invention may relate to a microarray chip comprising pooled probe set 13 to detect chromosomal abnormality in copy number of Chromosome 13. The present invention may relate to a microarray chip comprising pooled probe set 14 to detect chromosomal abnormality in copy number of Chromosome 14. The present invention may relate to a microarray chip comprising pooled probe set 15 to detect chromosomal abnormality in copy number of Chromosome 15. The present invention may relate to a microarray chip comprising pooled probe set 16 to detect chromosomal abnormality in copy number of Chromosome 16. The present invention may relate to a microarray chip comprising pooled probe set 17 to detect chromosomal abnormality in copy number of Chromosome 17. The present invention may relate to a microarray chip comprising pooled probe set 18 to detect chromosomal abnormality in copy number of Chromosome 18. The present invention may relate to a microarray chip comprising pooled probe set 19 to detect chromosomal abnormality in copy number of Chromosome 19. The present invention may relate to a microarray chip comprising pooled probe set 20 to detect chromosomal abnormality in copy number of Chromosome 20. The present invention may relate to a microarray chip comprising pooled probe set 21 to detect chromosomal abnormality in copy number of Chromosome 21. The present invention may relate to a microarray chip comprising pooled probe set 22 to detect chromosomal abnormality in copy number of Chromosome 22. The present invention may relate to a microarray chip comprising pooled probe set 23 to detect chromosomal abnormality in copy number of Chromosome X. The present invention may relate to a microarray chip comprising pooled probe set 24 to detect chromosomal abnormality in copy number of Chromosome Y. The present invention may relate to a microarray chip comprising pooled probe sets 1 to 24 to detect chromosomal abnormalities in copy numbers of the whole chromosomes.


In another embodiment, the present invention relates to a microarray chip comprising one or more pooled probe sets selected from the group consisting of pooled probe sets 25 to 32 to detect micro-deletion of specific chromosomal regions corresponding to the used pooled probe set. More specifically, the present invention may relate to a microarray chip comprising pooled probe set 25 to detect micro-deletion of 4p16.3 of Chromosome 4. The present invention may relate to a microarray chip comprising pooled probe set 26 to detect micro-deletion of 5p15.2 of Chromosome 5. The present invention may relate to a microarray chip comprising pooled probe set 27 to detect micro-deletion of 7q11.2 of Chromosome 7. The present invention may relate to a microarray chip comprising pooled probe set 28 to detect micro-deletion of 15q11-15q13 of Chromosome 15. The present invention may relate to a microarray chip comprising pooled probe set 29 to detect micro-deletion of 17p13.3 of Chromosome 17. The present invention may relate to a microarray chip comprising pooled probe set 30 to detect micro-deletion of 17p11.2 of Chromosome 17. The present invention may relate to a microarray chip comprising pooled probe set 31 to detect micro-deletion of 22q11.2 of Chromosome 22. The present invention may relate to a microarray chip comprising pooled probe set 32 to detect micro-deletion of Xp22.31 of Chromosome X. The present invention may relate to a microarray chip comprising pooled probe sets 25 to 32 to detect micro-deletions in specific chromosomal regions associated with Wolf-Hirschhorn, Cri-Du-Chat, William, Prader-willi, Angelman, Miller-Dieker Lissencephaly, Smith-Magenis, Digeorge, or Steroid Sulfatase Deficiency syndrome.


In another embodiment, the microarray chip may comprise pooled probe sets 1 to 24, and further comprise one or more pooled probe sets selected from the group consisting of pooled probe sets 25 to 32 to simultaneously detect chromosomal abnormality in copy number and micro-deletion of a specific chromosomal region.


In an embodiment of the present invention, all probes belonging to each pooled probe set are mixed and immobilized in a spot on the substrate, one spot comprises only one pooled probe set, and the microarray chip may comprise at least one spot for each pooled probe set. The number of the spots for each pooled probe set may be properly adjusted by considering the intended use and purpose, the total number of the used pooled primer sets, the total area of the chip, and the like, preferably 1- to 20-fold, more preferably 1- to 10-fold, but not limited thereto. The concentration of the immobilized pooled probe set may be 2 to100 pg per a spot. The shape of spot may be circular with 50 to 500 um of diameter, and the interval between the centers of the two adjacent spots may be more than the sum of the radiuses of the two spots, preferably 10 to 1000 um, but not limited thereto. The size and density of spot can be adjusted suitably depending upon the intended resolution of the analyzing system for the microarray.


The substrate of the microarray chip may be any one which is widely used in the art. Preferably, the substrate may have a functional group for immobilizing the probe on its surface, or be made from material being capable of forming three-dimensional structure. For example, the substrate may be made one or more materials selected from the group consisting of silicone wafer, glass, polycarbonate, nitrocellulose or nylon membrane, polymer films such as polystyrene or polyurethane, and porous materials.


The pooled probe sets 1 to 32 are summarized in following Tables 2 to 33, respectively.









TABLE 2







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 1 (Pooled probe set 1).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC27_N16
1
1p36.13
19285054
19395702
110648
27.05
perfect


BAC25_C19
1
1p34.3
37170820
37285410
114590
27.03
perfect


BAC153_I07
1
1p32.3
54534001
54652201
118200
25.17
perfect


BAC217_C19
1
1p31.3
66822471
66925991
103520
29.12
perfect


BAC59_D13
1
1p31.1
82036545
82127504
90959
16.89
perfect


BAC54_I02
1
1p22.2
88351055
88477939
126884
27.41
perfect


BAC163_C09
1
1p21.3
98212449
98304737
92288
19.25
perfect


BAC218_G03
1
1p13.3
110490903
110607934
117031
28.98
perfect


BAC152_F22
1
1p12
117910281
118017185
106904
31.56
perfect


BAC34_P03
1
1q21.1
145406431
145498341
91910
40.7
perfect


BAC36_I16
1
1q22
154638024
154757662
119638
28.64
perfect


BAC145_L11
1
1q23.3
162863145
162984456
121311
18.36
perfect


BAC37_O23
1
1q25.1
173728216
173843014
114798
27.28
perfect


BAC239_G19
1
1q25.3
182629151
182749008
119857
34.15
perfect


BAC105_P13
1
1q31.3
197091253
197202904
111651
29.21
perfect


BAC57_N17
1
1q41
212185136
212282511
97375
12.14
perfect


BAC239_A12
1
1q42.2
230646993
230768530
121537
23.07
perfect


BAC171_H09
1
1q43
237712318
237831818
119500
37.74
perfect


BAC222_E02
1
1q43-
241619770
241732398
112628
25.61
perfect




1q44
















TABLE 3







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 2 (Pooled probe set 2).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC126_E04
2
2p24.1
23602292
23712473
110181
26.88
perfect


BAC197_E10
2
2p23.3
27095066
27214934
119868
34.22
perfect


BAC43_A02
2
2p22.2
36561484
36662948
101464
20.26
perfect


BAC33_C05
2
2p21
47070526
47183203
112677
24.72
perfect


BAC59_D21
2
2p16.2
54644967
54759707
114740
17.3
perfect


BAC12_G01
2
2p14
66500267
66617870
117603
14.47
perfect


BAC141_F07
2
2p12
80058556
80173059
114503
30.95
perfect


BAC163_C22
2
2q11.2
98459189
98579536
120347
26.6
cutoff


BAC36_H22
2
2q12.1
104773145
104878334
105189
19.15
perfect


BAC143_G24
2
2q14.2
119233409
119344014
110605
23.45
perfect


BAC238_G01
2
2q21.3
134822967
134934479
111512
24.36
perfect


BAC252_A16
2
2q22.3
144992590
145107231
114641
25.6
perfect


BAC46_J12
2
2q23.3
152925448
153050963
125515
27.5
perfect


BAC57_C12
2
2q24.2
162502518
162618183
115665
21.69
perfect


BAC34_F17
2
2q31.1
176602023
176707880
105857
23.81
perfect


BAC79_L21
2
2q32.1
188831021
188946517
115496
38.48
cutoff


BAC39_M07
2
2q33.1
199897389
200006981
109592
23.1
perfect


BAC156_K09
2
2q34
213472534
213593503
120969
23.99
perfect


BAC195_I06
2
2q36.3
227821772
227932448
110676
31.33
cutoff


BAC88_K20
2
2q37.2
235531135
235632693
101558
24.89
perfect
















TABLE 4







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 3 (Pooled probe set 3).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC197_B21
3
3p26.1
7619617
7724613
104996
27.53
perfect


BAC158_C03
3
3p24.2
25484129
25595025
110896
28.09
perfect


BAC144_C11
3
3p22.3
35638825
35740337
101512
14.74
perfect


BAC186_N05
3
3p21.1
53696599
53834500
137901
17.93
perfect


BAC103_F06
3
3p14.2
62398546
62502148
103602
21.41
perfect


BAC114_B23
3
3p13
73635347
73736476
101129
25.76
perfect


BAC102_E23
3
3p12.1
85609865
85716578
106713
27.09
cutoff


BAC119_G21
3
3q12.1
101052953
101162681
109728
27.18
perfect


BAC68_H20
3
3q21.3
128151883
128252811
100928
21.7
perfect


BAC237_M11
3
3q22.3
138963565
139067350
103785
37.46
perfect


BAC168_G04
3
3q24
148525900
148639469
113569
13.33
perfect


BAC61_M02
3
3q26.2
170262202
170373830
111628
19.11
perfect


BAC36_N19
3
3q27.2
187248391
187394849
146458
31.4
perfect
















TABLE 5







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 4 (Pooled probe set 4).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC60_H08
4
4p16.1
7720688
7861517
140829
29.89
perfect


BAC26_C10
4
4p15.2
23475540
23578343
102803
21.45
perfect


BAC68_O19
4
4p13
41292867
41404193
111326
22.43
perfect


BAC102_G08
4
4p12
48150595
48249718
99123
28.41
perfect


BAC127_B16
4
4q13.1
59583784
59686203
102419
46.85
perfect


BAC176_G14
4
4q13.3
73393978
73505886
111908
33
perfect


BAC41_O05
4
4q21.1
78338989
78446698
107709
32.37
perfect


BAC37_H04
4
4q22.3
97252146
97364557
112411
36.26
perfect


BAC115_C13
4
4q25
110284871
110404724
119853
32.8
perfect


BAC30_N21
4
4q26
120049222
120165673
116451
34.35
perfect


BAC220_D24
4
4q28.3
134266064
134345943
79879
17.21
perfect


BAC106_P17
4
4q31.23
148133315
148244415
111100
24.89
perfect


BAC41_O11
4
4q32.1
157831745
157956901
125156
33.17
perfect


BAC157_P10
4
4q32.3
169969985
170078568
108583
24.78
perfect


BAC27_L15
4
4q35.1
186746250
186847111
100861
17.97
perfect
















TABLE 6







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 5 (Pooled probe set 5).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC86_B20
5
5p15.2
14309256
14409481
100225
20.7
perfect


BAC33_N18
5
5p14.1
25999429
26158704
159275
50.82
perfect


BAC55_L24
5
5p13.2-
38403985
38527847
123862
32.3
perfect




5p13.1


BAC226_H03
5
5p12
44650743
44794555
143812
50.34
perfect


BAC156_E24
5
5q11.2
58505228
58611348
106120
28.98
perfect


BAC237_B02
5
5q13.2
71448934
71560365
111431
20.72
perfect


BAC29_D17
5
5q14.3
87983275
88080419
97144
13.38
perfect


BAC139_M23
5
5q22.2
112566458
112663325
96867
25.1
perfect


BAC21_J16
5
5q23.2
122456733
122564806
108073
18.34
perfect


BAC27_N23
5
5q31.1
134366901
134477059
110158
25.77
perfect


BAC148_D23
5
5q33.1
149559660
149666803
107143
33.2
perfect


BAC186_L21
5
5q33.3
158095591
158197200
101609
21.95
perfect


BAC238_E21
5
5q34
166902727
167015224
112497
26.28
perfect


BAC175_N07
5
5q35.2
173228824
173344604
115780
32.25
perfect
















TABLE 7







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 6 (Pooled probe set 6).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC125_G09
6
6p22.3
15531954
15630750
98796
18.2
perfect


BAC182_E20
6
6p21.31
33763536
33871714
108178
23.32
perfect


BAC81_C08
6
6p21.1
44089494
44207145
117651
36.03
perfect


BAC24_P12
6
6p12.1
56485318
56604851
119533
24.39
perfect


BAC76_A23
6
6q13
70921840
71025599
103759
23.1
perfect


BAC26_F16
6
6q14.1
80924435
81042966
118531
38.26
perfect


BAC43_M14
6
6q15
91854172
91954978
100806
26.81
perfect


BAC27_P17
6
6q21
107961222
108059168
97946
16.28
perfect


BAC1_N23
6
6q22.2
118469623
118588188
118565
39.06
perfect


BAC247_D17
6
6q22.33
128420486
128521502
101016
26.06
perfect


BAC101_M04
6
6q23.3
138540130
138646539
106409
33.26
perfect


BAC90_F08
6
6q24.2
145084822
145186048
101226
27.88
perfect


BAC118_M18
6
6q25.3
157302386
157406286
103900
26.82
perfect


BAC179_N12
6
6q26
161982831
162089918
107087
28.06
perfect
















TABLE 8







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 7 (Pooled probe set 7).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC231_L03
7
7p21.3
8392407
8510938
118531
29.27
perfect


BAC82_L17
7
7p21.1
17592473
17703514
111041
44.84
perfect


BAC218_N01
7
7p15.2
27134934
27246285
111351
9.83
perfect


BAC5_A09
7
7p14.1
41971065
42072309
101244
18.63
perfect


BAC170_M16
7
7p11.2
55122494
55230463
107969
15.74
perfect


BAC119_K16
7
7q11.22
69481854
69594033
112179
25.95
perfect


BAC248_P06
7
7q21.11
81669117
81775391
106274
37.26
perfect


BAC96_F02
7
7q21.3
96395369
96489640
94271
18.65
perfect


BAC139_J04
7
7q31.1
107817605
107913546
95941
39.38
perfect


BAC76_K13
7
7q32.3
131748178
131844288
96110
19.98
perfect


BAC192_N04
7
7q33
136905463
137009239
103776
28.94
perfect


BAC154_A21
7
7q34
142687940
142795907
107967
30.5
cutoff


BAC120_I09
7
7q36.3
154894609
154994317
99708
23.61
perfect
















TABLE 9







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 8 (Pooled probe set 8).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC150_M15
8
8p23.1
10706703
10809056
102353
19.34
perfect


BAC149_J08
8
8p21.2
25832727
25937836
105109
19.1
perfect


BAC63_M21
8
8p12
32590447
32691100
100653
19.7
perfect


BAC147_O15
8
8p11.21
41666791
41781564
114773
32.27
perfect


BAC44_I16
8
8q11.21
49897613
49995230
97617
32.07
perfect


BAC30_N24
8
8q12.1
60026605
60149114
122509
21.26
perfect


BAC43_J01
8
8q13.2-
70570126
70680171
110045
28.01
perfect




8q13.3


BAC234_M17
8
8q21.11
77830466
77927688
97222
18.15
perfect


BAC68_K11
8
8q21.3
89314328
89429821
115493
36.14
perfect


BAC200_C08
8
8q22.3
103287335
103402138
114803
34.63
perfect


BAC237_M08
8
8q23.1
106578808
106677714
98906
22.33
perfect


BAC61_N10
8
8q23.3
115840828
115937681
96853
55.69
perfect


BAC80_H19
8
8q24.13
125618138
125727013
108875
32.26
perfect


BAC150_P12
8
8q24.22
135673467
135809603
136136
44.66
perfect


BAC66_I02
8
8q24.3
140926132
141036056
109924
31.05
perfect
















TABLE 10







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 9 (Pooled probe set 9).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC80_F23
9
9p24.1
8308622
8413950
105328
26.68
perfect


BAC28_L14
9
9p22.3
14200117
14314787
114670
16.8
perfect


BAC137_L16
9
9p21.3
23665603
23759045
93442
15.87
perfect


BAC161_C10
9
9q21.11
70573449
70692423
118974
34.73
perfect


BAC92_D01
9
9q21.13
77918661
78033112
114451
25.64
perfect


BAC163_H11
9
9q21.33
86735179
86840533
105354
28.08
perfect


BAC12_E22
9
9q22.32
96781575
96901045
119470
19.94
perfect


BAC172_D10
9
9q31.2
108625127
108739308
114181
30.85
perfect


BAC149_L08
9
9q32
115391007
115517471
126464
45.78
perfect


BAC188_O18
9
9q33.2
123482469
123582132
99663
24.2
cutoff


BAC126_N07
9
9q34.13
133669938
133789283
119345
38.25
perfect
















TABLE 11







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 10 (Pooled probe set 10).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC170_F05
10
10p15.1
6507791
6616487
108696
34.33
perfect


BAC102_J19
10
10p14
11202864
11309078
106214
19.15
perfect


BAC40_P04
10
10p13
13767514
13888447
120933
35.21
perfect


BAC141_E23
10
10p12.1
24780160
24883204
103044
31.14
perfect


BAC246_I22
10
10p11.22-
34430488
34539131
108643
24.22
perfect




10p11.21


BAC14_K16
10
10q11.21
44702716
44811838
109122
32.14
perfect


BAC52_B14
10
10q22.2
75183312
75288876
105564
29.18
perfect


BAC158_C10
10
10q23.31
89877530
89994800
117270
34.92
perfect


BAC155_O18
10
10q24.32
103344207
103439821
95614
24.62
perfect


BAC144_E19
10
10q25.2
114771881
114884798
112917
22.92
perfect


BAC218_E11
10
10q26.13
123194345
123298554
104209
28.5
perfect


BAC48_I12
10
10q26.13
126688077
126783871
95794
18.83
perfect


BAC182_N07
10
10q26.3
130655243
130754961
99718
19.47
perfect
















TABLE 12







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 11 (Pooled probe set 11).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC68_K10
11
11p15.2
12761489
12858699
97210
21.81
perfect


BAC90_E18
11
11p14.3
23422886
23527116
104230
52.57
perfect


BAC24_K17
11
11p13
34043974
34159762
115788
32.53
cutoff


BAC58_O19
11
11p11.2
45844027
45945118
101091
28.31
cutoff


BAC36_K05
11
11q12.2
61222569
61318879
96310
23.4
perfect


BAC150_P20
11
11q13.3
70323804
70440259
116455
27.67
perfect


BAC154_H22
11
11q14.1
80897410
80995179
97769
41.22
perfect


BAC26_C09
11
11q21
95341253
95452490
111237
33.28
perfect


BAC119_O13
11
11q22.3
105421723
105552485
130762
51.39
perfect


BAC195_O14
11
11q23.2
114533244
114632257
99013
9.77
perfect


BAC73_E17
11
11q23.3
120257418
120365515
108097
29.74
perfect


BAC142_K09
11
11q24.3
128061292
128178954
117662
22.6
perfect


BAC65_D19
11
11q25
132481572
132589407
107835
24.68
perfect
















TABLE 13







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 12 (Pooled probe set 12).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC60_I23
12
12p13.2
11701743
11809262
107519
24.36
perfect


BAC121_P21
12
12p11.23
26689788
26798168
108380
34.03
cutoff


BAC199_G02
12
12q12
39582488
39679490
97002
25.88
perfect


BAC65_G10
12
12q13.13
51215952
51320650
104698
29.86
perfect


BAC41_I18
12
12q13.2
53220091
53326596
106505
32.93
perfect


BAC10_M07
12
12q14.3
64499036
64596568
97532
24.53
perfect


BAC39_O14
12
12q21.1
73748137
73850966
102829
26.13
perfect


BAC144_K11
12
12q21.32
86754102
86851280
97178
45.83
perfect


BAC178_M15
12
12q23.2
101295480
101408773
113293
29.29
perfect


BAC134_M17
12
12q24.21
114892621
114996460
103839
27.37
perfect


BAC65_I21
12
12q24.31
122960995
123070617
109622
27.37
perfect


BAC27_E08
12
12q24.33
130071553
130182033
110480
33.15
cutoff
















TABLE 14







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 13 (Pooled probe set 13).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC28_H21
13
13q12.13
25143222
25245331
102109
27.22
perfect


BAC163_F01
13
13q12.3
30534492
30645395
110903
29.45
perfect


BAC78_C21
13
13q14.3
51185202
51293792
108590
31.63
perfect


BAC135_O03
13
13q21.31
61708361
61820641
112280
48.99
perfect


BAC237_P24
13
13q21.33
71970035
72071453
101418
28.19
perfect


BAC84_N09
13
13q31.1
85224563
85327840
103277
31.28
perfect


BAC8_C18
13
13q32.1
96626342
96733503
107161
30.97
perfect


BAC133_G23
13
13q33.3
105875452
105978338
102886
21.77
perfect


BAC116_B15
13
13q34
109618954
109723753
104799
17.82
perfect
















TABLE 15







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 14 (Pooled probe set 14).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC236_F24
14
14q11.2
22882169
22975980
93811
17.8
perfect


BAC22_E01
14
14q13.1
33051075
33164524
113449
19.32
perfect


BAC37_K09
14
14q21.3
46529853
46639288
109435
27.08
perfect


BAC79_J20
14
14q23.1
56316365
56426133
109768
29.93
perfect


BAC50_I09
14
14q23.1
58784060
58905688
121628
24.33
perfect


BAC15_E12
14
14q24.2
69499442
69615359
115917
28.53
perfect


BAC63_O11
14
14q24.3
76716134
76840351
124217
32.66
perfect


BAC11_N10
14
14q31.3
85117915
85219573
101658
29.18
perfect


BAC39_P02
14
14q32.13
93445004
93557400
112396
35.57
perfect


BAC101_O15
14
14q32.31
100503542
100635568
132026
27.01
perfect
















TABLE 16







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 15 (Pooled probe set 15).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC66_K21
15
15q14
34891569
34992991
101422
25.7
perfect


BAC162_K11
15
15q15.1
39911516
40017191
105675
21.32
cutoff


BAC178_K16
15
15q21.1
46636419
46734018
97599
25.1
perfect


BAC21_K13
15
15q21.3
55514115
55623430
109315
31.83
perfect


BAC167_M02
15
15q22.2
60804474
60910289
105815
24.56
perfect


BAC88_F18
15
15q22.33
65152593
65266241
113648
30.99
perfect


BAC168_F12
15
15q24.1
72269733
72378984
109251
30.64
perfect


BAC10_E08
15
15q25.1
78883923
78994040
110117
30.34
perfect


BAC177_H09
15
15q25.3
86262818
86362227
99409
24.84
perfect


BAC41_K03
15
15q26.2
94625429
94735125
109696
19.8
perfect
















TABLE 17







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 16 (Pooled probe set 16).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC38_I04
16
16p13.2
7236659
7345065
108406
32.18
perfect


BAC96_J19
16
16p12.3
17301008
17430114
129106
46.81
perfect


BAC120_K24
16
16p12.1
22880407
23000700
120293
47.38


BAC177_P23
16
16p11.2
27677745
27824753
147008
50.93
perfect


BAC247_B03
16
16q12.1
46582236
46708479
126243
41.47
perfect


BAC117_H14
16
16q13
54857795
54968957
111162
30.04
perfect


BAC96_G02
16
16q21
63766221
63897034
130813
45.04
perfect


BAC24_D17
16
16q22.3
71392858
71500676
107818
19.52
perfect


BAC223_D19
16
16q23.1-
78124140
78223124
98984
22.27
perfect




16q23.2
















TABLE 18







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 17 (Pooled probe set 17).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC200_M05
17
17p13.1
9003380
9112032
108652
33.57
perfect


BAC50_A03
17
17p12
13241293
13370269
128976
38.5
cutoff


BAC149_H11
17
17q11.2
23828734
23932422
103688
30.88
cutoff


BAC29_G13
17
17q12
34946421
35061427
115006
33.07
perfect


BAC238_E06
17
17q21.33
45499528
45605085
105557
31.58
perfect


BAC150_O15
17
17q22
53010880
53111488
100608
24.52
perfect


BAC70_P11
17
17q24.2
61859026
61963885
104859
32.08
perfect


BAC70_N11
17
17q24.3
66884538
66990764
106226
27.85
perfect


BAC116_E10
17
17q25.1
70287026
70396381
109355
22.68
perfect


BAC48_K14
17
17q25.3
74796956
74899041
102085
25.69
perfect
















TABLE 19







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 18 (Pooled probe set 18).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC57_H08
18
18p11.31
6965630
7083991
118361
34.11
perfect


BAC141_I04
18
18p11.21
13204635
13303487
98852
27.8
perfect


BAC252_H16
18
18q12.1
23925558
24030666
105108
27.58
perfect


BAC232_E19
18
18q12.2
33328623
33432257
103634
20
perfect


BAC149_I18
18
18q12.3
40492533
40595058
102525
22.9
perfect


BAC186_P19
18
18q21.1
45732647
45836159
103512
26.99
perfect


BAC151_L02
18
18q21.31
52732610
52834222
101612
23.29
perfect


BAC230_C11
18
18q21.33
59076118
59172880
96762
34.04
perfect


BAC43_A24
18
18q22.3
66979772
67088762
108990
33.25
perfect


BAC184_J04
18
18q23
72199921
72294263
94342
15.35
perfect
















TABLE 20







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 19 (Pooled probe set 19).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC178_L22
19
19p13.2
7576484
7669091
92607
37.74
perfect


BAC160_C11
19
19p13.13
12904676
13008809
104133
38.79
perfect


BAC131_N13
19
19p13.11
19434885
19546124
111239
31.54
perfect


BAC54_N22
19
19q12
33062070
33154105
92035
40.36
perfect


BAC233_K14
19
19q12
36444841
36543930
99089
14.4
perfect


BAC162_K04
19
19q13.2
44526488
44654105
127617
42.27
perfect


BAC76_E22
19
19q13.32
52164544
52276406
111862
48.89
perfect


BAC211_B15
19
19q13.33
54533050
54646094
113044
43.14
perfect


BAC101_H02
19
19q13.42
60824176
60920115
95939
35.56
cutoff


BAC193_C07
19
19q13.42
61263496
61377134
113638
49.1
perfect
















TABLE 21







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 20 (Pooled probe set 20).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC247_K09
20
20p12.2
10566085
10662978
96893
25.39
perfect


BAC26_J24
20
20p12.1
16274790
16388873
114083
29.8
perfect


BAC75_H16
20
20p11.23
20305001
20413595
108594
26.25
perfect


BAC37_M13
20
20p11.21
24534588
24661098
126510
40.05
perfect


BAC19_G17
20
20q11.22
33307625
33429006
121381
38.42
perfect


BAC82_B07
20
20q12
38733854
38837009
103155
30.96
perfect


BAC96_H08
20
20q13.13
48537102
48674679
137577
38.72
perfect


BAC166_J02
20
20q13.2
51088180
51201285
113105
36.98
perfect


BAC41_E11
20
20q13.32
56785564
56882008
96444
19.43
perfect


BAC146_N07
20
20q13.33
59644343
59742642
98299
24.11
perfect
















TABLE 22







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 21 (Pooled probe set 21).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC102_F10
21
21q11.2
14571029
14713371
142342
42.45
perfect


BAC240_M07
21
21q21.1
18530589
18634646
104057
26.14
perfect


BAC200_O02
21
21q21.1
21711102
21810394
99292
32.67
perfect


BAC97_O19
21
21q21.2
25370877
25503818
132941
39.84
perfect


BAC119_K07
21
21q21.3
27746038
27845927
99889
37.68
perfect


BAC200_A23
21
21q21.3
30135569
30259474
123905
34.86
perfect


BAC221_D22
21
21q22.11
32955508
33054881
99373
36.91
perfect


BAC100_D11
21
21q22.12
35144569
35254293
109724
22.05
perfect


BAC33_D15
21
21q22.13
38404833
38548664
143831
46.69
perfect


BAC126_M10
21
21q22.3
42518138
42622787
104649
25.5
perfect
















TABLE 23







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome 22 (Pooled probe set 22).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC169_G07
22
22q11.22-
21785657
21898492
112835
36.87
perfect




22q11.23


BAC153_I19
22
22q12.1
24588271
24689820
101549
42.87
perfect


BAC100_P10
22
22q12.1
26700102
26828418
128316
35.55
perfect


BAC37_J03
22
22q12.2
28383741
28496470
112729
34.07
perfect


BAC187_K08
22
22q12.3
31516566
31610860
94294
31.84
perfect


BAC131_H09
22
22q12.3
35000557
35127809
127252
35.12
perfect


BAC106_C07
22
22q13.1
36052892
36157674
104782
30.88
perfect


BAC66_M06
22
22q13.1
39087833
39203708
115875
44.27
perfect


BAC51_M21
22
22q13.2
41943878
42052803
108925
26.49
perfect


BAC153_O04
22
22q13.31
45111661
45224294
112633
32.1
perfect
















TABLE 24







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome X (Pooled probe set 23).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC70_N16
X
Xp22.2
12242131
12358309
116178
31.61
perfect


BAC22_H14
X
Xp22.12
19530397
19656785
126388
32.17
perfect


BAC65_L14
X
Xp21.2
29622036
29749045
127009
37.56
perfect


BAC151_A03
X
Xp11.4
39814492
39933265
118773
25.85
perfect


BAC49_G05
X
Xp11.23
48447858
48563861
116003
46.6
cutoff


BAC130_K20
X
Xq11.1
62871569
62971143
99574
58.25
perfect


BAC103_N15
X
Xq13.1
71429226
71542407
113181
54.37
perfect


BAC136_M01
X
Xq21.1
79808105
79900354
92249
40.85
perfect


BAC6_B17
X
Xq21.33
96022346
96133479
111133
36.07
perfect


BAC141_P03
X
Xq22.2-
103499406
103603159
103753
54.93
perfect




Xq22.3


BAC246_K02
X
Xq22.3
106558477
106677904
119427
35.9
perfect


BAC91_J24
X
Xq22.3
110215405
110332259
116854
29.71
perfect


BAC97_C11
X
Xq25
122558528
122669264
110736
42.3
perfect


BAC63_G23
X
Xq25
128963506
129081155
117649
35.44
perfect


BAC73_B07
X
Xq26.2
131631150
131750852
119702
34.86
perfect


BAC162_B10
X
Xq27.2
141489490
141600695
111205
52
perfect


BAC119_C15
X
Xq28
149354893
149476738
121845
37.97
perfect
















TABLE 25







A pooled probe set specific to detecting chromosomal abnormality


in copy number of Chromosome Y (Pooled probe set 24).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Megablast

















BAC24_K23
Y
Yp11.2
8029316
8132309
102993
61.88
cutoff


BAC205_L13
Y
Yp11.2
8607008
8706082
99074
19.28
perfect


BAC127_H21
Y
Yq11.21
13495385
13609582
114197
62.45
perfect


BAC192_M14
Y
Yq11.21
13970303
14064396
94093
63.95
cutoff


BAC101_I21
Y
Yq11.21
14086977
14174195
87218
79.82
perfect


BAC140_H17
Y
Yq11.221
16177603
16271634
94031
54.31
cutoff


BAC65_J16
Y
Yq11.221
17261230
17382688
121458
60.27
perfect


BAC180_K16
Y
Yq11.221
17415806
17520345
104539
63.58
perfect


BAC102_F03
Y
Yq11.221
17655546
17754959
99413
47.54
perfect


BAC31_L01
Y
Yq11.222
19643980
19738325
94345
72.53
perfect


BAC240_H05
Y
Yq11.222
20119719
20208748
89029
60.43
perfect
















TABLE 26







A pooled probe set specific to Wolf-Hirschhorn syndrome (WHS) (Pooled probe set 25).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Disease

















BAC50_H08
4
4p16.3
1784731
1916874
132143
47
WHS


BAC67_I12
4
4p16.3
1873050
1980658
107608
41
WHS


BAC100_E03
4
4p16.3
1878656
1996081
117425
46
WHS


BAC1_F06
4
4p16.3
1890838
1985011
94173
40
WHS


BAC135_O20
4
4p16.3
1932272
2020557
88285
57
WHS


BAC153_J14
4
4p16.3
2049082
2129247
80165
52
WHS
















TABLE 27







A pooled probe set specific to Cri-Du-Chat syndrome (CRI-DU-CHAT) (Pooled probe set 26).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Disease

















BAC143_N22
5
5p15.2
9511494
9591939
80445
25
CRI-DU-CHAT


BAC206_I13
5
5p15.2
9591934
9697824
105890
46
CRI-DU-CHAT


BAC252_N08
5
5p15.2
9710410
9842377
131967
49
CRI-DU-CHAT


BAC64_P22
5
5p15.2
9737985
9835999
98014
46
CRI-DU-CHAT


BAC208_N21
5
5p15.2
9741001
9872689
131688
46
CRI-DU-CHAT


BAC200_E05
5
5p15.2
9761722
9855167
93445
48
CRI-DU-CHAT


BAC240_K06
5
5p15.2
9775708
9856013
80305
48
CRI-DU-CHAT
















TABLE 28







A pooled probe set specific to William syndrome (WBS) (Pooled probe set 27).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Disease

















BAC69_O08
7
7q11.23
72999634
73099848
100214
50
WBS


BAC66_N22
7
7q11.23
73117049
73203244
86195
69
WBS


BAC180_N24
7
7q11.23
73146401
73238493
92092
71
WBS


BAC67_C05
7
7q11.23
73215245
73346881
131636
54
WBS


BAC183_A12
7
7q11.23
73343227
73447186
103959
63
WBS


BAC123_D05
7
7q11.23
73347015
73426987
79972
64
WBS
















TABLE 29







A pooled probe set specific to Prader-willi syndrome (PWS)


and Angelman syndrome (AS) (Pooled probe set 28).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Disease

















BAC188_N24
15
15q11.2
21607011
21719880
112869
59
PWS


BAC223_H02
15
15q11.2
22694476
22779891
85415
57
PWS


BAC217_F02
15
15q11.2
22767885
22856153
88268
33
PWS


BAC71_A18
15
15q11.2
23101822
23188315
86493
49
PWS


BAC5_L18
15
15q11.2-
23211569
23311228
99659
50
PWS




15q12


BAC248_C13
15
15q12
24281096
24383877
102781
50
PWS


BAC78_F07
15
15q12
24369659
24485628
115969
45
PWS


BAC180_J22
15
15q12
24398126
24505662
107536
47
PWS


BAC21_O06
15
15q12
24429411
24553835
124424
42
PWS


BAC105_L07
15
15q12
24456681
24577646
120965
38
PWS
















TABLE 30







A pooled probe set specific to Miller-Dieker Lissencephaly


syndrome (MDLS) (Pooled probe set 29).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Disease

















BAC95_J10
17
17p13.3
26911
101177
74266
35
MDLS


BAC75_C17
17
17p13.3
138128
251064
112936
40
MDLS


BAC110_O13
17
17p13.3
157851
293897
136046
49
MDLS


BAC63_J08
17
17p13.3
2340899
2445465
104566
69
MDLS


BAC190_F10
17
17p13.3
2388499
2488318
99819
66
MDLS


BAC186_M15
17
17p13.3
2460766
2633700
172934
53
MDLS


BAC183_M06
17
17p13.3
2489245
2574271
85026
42
MDLS


BAC135_N07
17
17p13.3
2510283
2624227
113944
47
MDLS


BAC148_F06
17
17p13.3
2517812
2603600
85788
41
MDLS


BAC31_H03
17
17p13.3
2517823
2603600
85777
41
MDLS
















TABLE 31







A pooled probe set specific to Smith-Magenis syndrome (SMS) (Pooled probe set 30).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Disease





BAC249_G12
17
17p11.2
17516703
17630050
113347
32
SMS


BAC41_D18
17
17p11.2
17630085
17765348
135263
28
SMS


BAC186_E14
17
17p11.2
18072869
18192554
119685
36
SMS
















TABLE 32







A pooled probe set specific to Digeorge syndrome (DGS) (Pooled probe set 31).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Disease

















BAC124_E21
22
22q11.21
17410620
17526065
115445
48
DGS


BAC196_A22
22
22q11.21
17446169
17542727
96558
48
DGS


BAC69_P21
22
22q11.21
17647712
17740090
92378
69
DGS


BAC141_K20
22
22q11.21
17690557
17773366
82809
53
DGS


BAC169_K21
22
22q11.21
17690557
17773305
82748
53
DGS


BAC145_P12
22
22q11.21
17726605
17889246
162641
43
DGS


BAC224_F10
22
22q11.21
17759912
17870524
110612
43
DGS
















TABLE 33







A pooled probe set specific to Steroid Sulfatase deficiency (STS) (Pooled probe set 32).














Bac_ID
Chr. No.
Cyto
Bac_start
Bac_end
Bac_size
Repeat %
Disease

















BAC221_A12
X
Xp22.31
7088532
7186942
98410
46
STS


BAC191_E24
X
Xp22.31
7397582
7504424
106842
44
STS


BAC231_F19
X
Xp22.31
7547752
7636445
88693
42
STS









The microarray according to the present invention may be an array comparative genome hybridization (aCGH)-based in vitro diagnostic microarray. CGH has been commonly used to detect an amplification or micro-deletion of a specific chromosomal region. Recently, it has been combined with DNA microarray technology (i.e., aCGH), making it possible to analyze a large scale of DNA at one time. Further, the aCGH technology also makes it possible to simultaneously detect change in the gene expression amount and aberration in DNA copy number.


In another aspect, the present invention relates to a method of preparing the microarray chip comprising the step of immobilizing one or more selected from the group consisting of pooled probe sets 1 to 32 in spots on a substrate, wherein all probes belonging to each pooled probe set are immobilized together in a spot, and the microarray chip may comprise at least one spot for each pooled probe set. The number of the spots for each pooled probe set may be properly adjusted by considering the intended use and purpose, the total number of the used pooled primer sets, the total area of the chip, and the like.


The microarray may be prepared by the general method in the art. For example, the probe is immobilized on a substrate for microarray through physical or chemical binding. The probe may be immobilized according to the general immobilization method used in the preparation of the microarray chip, for examples photolithography, piezoelectric printing, micro-pipetting method or spotting method.


As described above, the substrate may be made one or more materials selected from the group consisting of silicone wafer, glass, polycarbonate, nitrocellulose or nylon membrane, polymer films such as polystyrene or polyurethane, and porous materials.


In yet another aspect embodiment, the present invention relates to a method of detecting chromosomal abnormalities using the microarray chip comprising one or more selected from the group consisting of pooled probe sets 1 to 32. The chromosomal abnormalities may include copy number variation(s) and/or micro-deletion(s) of specific chromosomal region(s) associated with various genetic alterations including pre-natal and/or post-natal disorders. Therefore, the method of the present invention may be used prenatally as well as postnatally. For detection by the present invention, micro well plate, for example 96-well plate may be used so that labeling, hybridization, and washing process can be performed with the automatic machines (for examples, Biomek, and Genetix robot).


More specifically, the method of detecting chromosomal abnormalities may comprise the steps of:


providing a microarray chip by immobilizing one or more selected from the group consisting of pooled probe sets 1 to 32 in spots on a substrate as described above;


labeling a test sample DNA and a reference DNA with different labels from each other;


fragmentizing the labeled DNAs, and applying the obtained test sample DNA fragments and the obtained reference DNA fragments onto the spots on the microarray chip, respectively, to hybridize the DNA fragments with the probes in the spot;


measuring a signal intensity from each pooled probe set hybridized with the test sample DNA or the reference DNA; and


comparing the signal intensity from the test sample DNA over that from the reference DNA.


The signal intensity indicates the hybridization ratio of the test sample or reference DNA fragment. The result of the comparison provides the bases of determination of chromosomal abnormality, as follows:


No chromosomal abnormality, if the hybridization ratio of the target genomic DNA is the same as that of the reference DNA is decided to produce no chromosome abnormality,


Chromosome amplification, if the hybridization ratio of the target genomic DNA is higher than that of the reference DNA.


Chromosome deletion, if the hybridization ratio of the target genomic DNA is lower than that of the reference DNA.


The labels used in the labeling step for the test sample DNA and the reference DNA may be fluorescent dyes with different colors from each other and independently selected from the group consisting of radioactive isotope, fluorescent material, chemical luminescent, and enzyme. For examples, the labels are selected from the group consisting of 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, bodipy, Texas red, FITC (Fluorescein Isothiocyanate), rhodamine, d-NTP (including d-UTP), reactive dye having amino-allyl modified dNTPs, horseradish peroxidase, biotin and etc. In the concrete embodiment of the present invention, the test sample DNA may be labeled with Cy3 (green fluorescence), and the reference DNA with Cy5 (red fluorescence). Then, the two fluorescently hybridized intensities from each probe are captured by a proper image analyzer, such as, a fluorescent image scanner. The observed two fluorescently hybridized intensities may be converted into values of copy numbers by a proper analysis software, such as MacView™ (Macrogen). The ratio of the value from the test sample over that from the reference shows whether the rest sample has any changes in copy number compared to the reference. The copy number may be a copy number of a specific chromosome, or a specific chromosomal region, which can be detected by the used probe.


Specifically, after co-hybridization of labeled test sample and reference DNA fragment performed, the hybridization ratios of the probes and the test sample or reference DNA fragment may be represented as T/R which is the value of sample (T)'s fluorescence intensity divided by reference (R)'s fluorescence intensity. Theoretically T/R may equal 1, if the test sample and the reference have the same number of chromosome copies (n=2, disomic state). Therefore, T/R>1 means that the sample's fluorescence intensity is higher than the reference, indicating the sample's chromosome or chromosomal region corresponding to the used probe is amplified (copy number gain). In contrast, T/R<1 means that the sample's fluorescence intensity is lower than the reference, indicating the sample's chromosome or chromosomal region corresponding to the used probe is deleted (copy number loss).


Even though chromosomes have the same copy numbers (i.e., two copy of each chromosome for test and reference samples), each clone's T/R value is slightly different due to the inherent genomic polymorphism compounded by the experimental noise. Therefore, to ascertain the meaningful and measurable copy number variations as output, an empirically determined average value of log 2(T/R) of each clone was used in the examples of the present invention.


Generally, it is difficult to distinguish between three-time amplification (trisomic state) and two-time amplification (disomic state) in the copy number. When a normal DNA is used as the reference DNA, it may be difficult to exactly distinguish the trisomy of Chromosome X in karyotype 47, XXX (super female syndrome) form normal state (karyotype 46, XY). Therefore, it may be preferable to use a DNA sample of Kleinfelter syndrome, karyotype 47, XXY, as a reference, when detecting the copy number aberration in Chromosome X.


The test sample DNA may be extracted from amniotic fluid, peripheral blood, chorionic villus, umbilical cord blood, placenta villi, and cultured cells obtained from a subject. The subject may be a mammalian, preferably human, and more preferably fetus, newborn infants (until 4-weeks after birth) or children.


In yet another embodiment, the present invention provides a kit for diagnosing a disease associated with a chromosomal abnormality, comprising the microarray chip as described above.


In yet another embodiment, the present invention provides a method of diagnosing a disease associated with a chromosomal abnormality using the microarray chip, comprising the steps of:


providing a microarray chip by immobilizing one or more selected from the group consisting of pooled probe sets 1 to 32 in spots on a substrate as described above;


labeling a test sample DNA from a patient and a reference DNA with different labels from each other;


fragmentizing the labeled DNAs, and applying the obtained test sample DNA fragments and the obtained reference DNA fragments onto the spots on the microarray chip, respectively, to hybridize the DNA fragments with the probes;


measuring a signal intensity from each probe hybridized with the test sample DNA or the reference DNA; and


comparing the signal intensity from the test sample DNA over that from the reference DNA, to determine that the patient has a disease associated with a chromosomal abnormality detectable by the used pooled probe set, when a difference is detected between the signal intensity from the test sample DNA and that from the reference DNA.


The test sample DNA may be extracted from amniotic fluid, peripheral blood, chorionic villus, umbilical cord blood, placenta villi, and cultured cells obtained from a patient. The patient may be a mammalian, preferably human, and more preferably fetus, newborns or children.


The disease may be one or more selected from the group consisting of Down syndrome, Patau syndrome, Edward syndrome, Tuner syndrome, Klinefelter syndrome, sex chromosome aneusomies (e.g., super female syndrome, super male syndrome, etc.), most frequently appearing micro-deletion syndromes (e.g., Wolf-Hirschhorn syndrome, Cri-Du-Chat syndrome, William syndrome, Prader-willi syndrome, Miller-Dieker Lissencephaly syndrome, Smith-Magenis syndrome, Digeorge syndrome, Steroid Sulfatase deficiency syndrome, etc.), and the like.


For example, a microarray chip comprising pooled probe set 21 is used and the value of (T/R) corresponding to the pooled probe set 21 immobilized spot is higher than 1, indicating that the subject has Down syndrome.


Therefore, in the kit or the method of the present invention, the microarray chip may comprise


at least pooled probe set 21 for diagnosing Down syndrome;


at least pooled probe set 13 for diagnosing Patau syndrome;


at least pooled probe set 18 for diagnosing Edward syndrome;


at least pooled probe sets 23 and 24 for diagnosing Tuner syndrome, Klinefelter syndrome, Super female syndrome, or Super male syndrome;


at least pooled probe set 25 for diagnosing Wolf-Hirschhorn syndrome;


at least pooled probe set 26 for diagnosing Cri-Du-Chat syndrome;


at least pooled probe set 27 for diagnosing William syndrome;


at least pooled probe set 28 for diagnosing Prader-willi syndrome and/or Angelman syndrome;


at least pooled probe set 29 for diagnosing Miller-Dieker Lissencephaly syndrome;


at least pooled probe set 30 for diagnosing Smith-Magenis syndrome;


at least pooled probe set 31 for diagnosing Digeorge syndrome; or


at least pooled probe set 32 for diagnosing Steroid Sulfatase deficiency syndrome.


Alternatively, the kit or the method of the present invention may use at least one selected from the group consisting of pooled probe sets 13, 18, 21, and 23 to 32 for simultaneously diagnosing at least one selected from the group consisting of Down syndrome, Patau syndrome, Edward syndrome, Tuner syndrome, Klinefelter syndrome, Super female syndrome, Super male syndrome, Wolf-Hirschhorn syndrome, Cri-Du-Chat syndrome, William syndrome, Prader-willi syndrome, Angelman syndrome, Miller-Dieker Lissencephaly syndrome, Smith-Magenis syndrome, Digeorge syndrome, and Steroid Sulfatase deficiency syndrome.


There are several benefits of the diagnosing kit and method, as follows: (1) the results are rapidly available, for example, within 36 hours after the sample collection in the case of amniocentesis, compared to 7-22 days for routine chromosome analysis; not only the most frequently appearing anuesomies, but also the most frequently observed microdeletions in mammalians, especially infants, can be obtain at one time by properly selecting and using the pooled probe sets of the present invention, whereby most time-saving and cost-effective comprehensive prenatal or postnatal evaluations can be achieved; the availability of the results obtained by the present invention along with consistent clinical information (i.e., fetal anomalies detected by karyotyping and ultrasonography) allows for more options that otherwise might not be available; and in the case of a culture failure when standard cytogenetic results are not available, accurate assessment on chromosome copy number for the most frequent aneusomies can be provided. More than two-thirds of all abnormalities can be identified by the present invention at the time of amniocentesis, and at least 90% of clinically significant chromosomal abnormalities detected in live-born infants.


The present invention is further explained in more detail with reference to the following examples. These examples, however, should not be interpreted as limiting the scope of the present invention in any manner.


Example 1

The following Examples generally refer to Korean Patent Application No. 2004-0066384 and U.S. patent application Ser. No. 11/211,185, which are incorporated herein as references.


Example 1
Preparation of KOGENOME 96,768 BAC Clones

1.1. Preparation of Genome Library


A genome was isolated from a Korean man, treated with HindIII enzyme and subjected to PFGE, to obtain a DNA fragment of about 100 Kb. The DNA fragment of the average size of 100 Kb was ligased to a BAC vector, and then, transformed into a host cell, E. coli (Competent cell, E. coli DH10B BAC). Herein, the E. coli cell line containing each DNA fragment was called as a clone, and 96,768 clones in total were obtained through the preparation of genome library.


1.1.1. Isolation of Genome


20 ml of the entire genomic DNA was isolated from semen of a Korean man, and subjected to a qualitative analysis and a quantitative analysis through agarose gel electrophoresis.


1.1.2. Fragmentation and Purification of Genome


The isolated genomic DNA was cleaved with BamHI, and subjected to PEG gel electrophoresis. The portion which was developed at the position of 100 Kb was collected, to isolate DNA fragments.


1.1.3. Transformation


The DNA fragments were inserted into BAC vectors (pECBAC1, Friijter et al. 1997) at BamHI site, and transformed into host cells (Competent cell, E. coli DH10B BAC). Then, the host cells were cultivated in a solid culture media to obtain clone of each cell. The clones were inoculated on 96-well format cell culture blot, and cultivated in a rotating incubator of 300 rpm at 37° C. for 18 hours.


The components of the used solid culture media are as follows:


LB (DIFCO, Cat. No 244620) 25 g


Pancreatic digest of casein 10 g


Yeast extract 5 g


Sodium chloride 10 g


Bacto Agar (DIFCO, Cat. No 214010) 15 g


Chroramphenicol (SIGMA, Cat. No. C0817) 13.6 mg


ddH2O Adjust to 1 L


DIFCO, BD, Sparks. MD21152


SIGMA, ST. louis, MI63178.


1.1.4. Preparation of Library Cell Stock Solution


25 μl of 65% glycerol was put into 384-well plate, and 25 μl of cells cultivated in 96-well format cell culture blot was added thereto (cells cultivated in four 96-well format cell culture blots were collected and stored in one 384-well plate). The top of the 384-well was sealed and stored at −70° C.


1.2. End-Sequencing


500 by of both ends of the 100 Kb DNA fragment were analyzed with a BAC vector specific primer, and the information for the inserted genomic DNA of each clone was confirmed and recorded.


1.2.1. Isolation of BAC DNA (Mini-Prep.)


1×LB 1 ml was put into 96-well culture blot, and library cell stock solution 1 ml was inoculated thereto. The cells stored in one 348-well plate were divisionally cultivated in four 96-well culture blots. The cells was cultivated in a rotating incubator of 300 rpm at 37° C. for 18 hours and, to prepare a sample stock solution in the amount of 25 ml per a well in five times. The remnant was centrifuged at 1000 rpm for 15 minutes, to obtain cells. DNA was collected by using a kit, Montage mini-prep.


1.2.2. Sequencing


Both ends of the DNA fragment inserted into each clone was treated with T7 and M13R primers, and the base sequences thereof were analyzed by using an Automatic DNA Sequencer, ABL 3700.


1.3. Bioinformatics


The analyzed base sequences were subjected to the sequence identity analysis compared with the base sequence of the entire human genome analyzed through the human genome project using a bioinformatics technology. Through the above processes, the position of the BAC DNA in genome of each clone in which the BAC DNA is inserted and the entire base sequence thereof were determined.


A blast search was performed by using the analyzed end sequence, and its position in the genome of each clone was determined by using the sequence identity analysis.


1.4. BAC Clone Identification by FISH


As described above, all of the clones were two end-sequenced using Applied Biosystems 3700 sequencers and their sequences were analyzed by BLAST and mapped according to their positions on the UCSC human genome database (http://www.genome.uscs.edu). Confirmation of locus specificity of about 4,500 clones was performed by removing multiple loci binding clones by individually examining by Fluorescent In Situ Hybridization (FISH). FISH was conducted by using the genomic DNA fragment contained in each BAC clone as a probe. In the FISH technique employing the DNA complementary binding principle, a probe which specifically binds to a specific region of a chromosome is directly bound to the chromosome fixed on a glass slide, to determine the position of the probe in the chromosome.


Example 2
Fabrication of Microarray Chip

2.1. Clone selection for Microarray Chip


350 clones shown in Tables 34 are selected for the microarry chip of the present invention as the followings:


(1) From the two end-sequenced and single locus FISH confirmed 4500 clones, 550 clones were chosen as the first set of clones.


(2) Insert size of at least 74-173 kb clones were chosen.


(3) For whole chromosome representing BAC clones, clones were chosen as a 12-15 Mb interval gap between two BAC clones.


(4) For microdeletion representing BAC clones, clones were selected to cover the region at a tiling pathway (i.e., no gap between the two adjacent clones).


(5) Copy Number Variation (CNV, i.e., within a normal human population, some regions of chromosomes are polymorphic) containing BAC clones were removed from the list and the neighboring clones were selected instead to more accurately detect the true copy number aberrations.


In addition, negative controls (non-hybridizing arabidopsis genomic DNA) was included in this chip.















TABLE 34







disease
Cyto.
OMIM_ID
gene/marker
clones





















Micro-
ANGELMAN SYNDROME
15q11-q13
105830
UBE3A



deletion
(same region deletion with PWS)



CRI-DU-CHAT SYNDROME
5p15.2
123450
TERT
7



DIGEORGE SYNDROME
22q11.2
188400
TBX1
7



MILLER-DIEKER
17p13.3
247200
LIS1
10



LISSENCEPHALY



SYNDROME



PRADER-WILLI SYNDROME
15q11-15q13
176270
SNRPN
10



SMITH-MAGENIS
17p11.2
182290
RAI1
3



SYNDROME



STEROID SULFATASE
Xp22.31
308100

3



DEFICIENCY DISEASE



WILLIAMS-BEUREN
7q11.2
194050
LIMK1
6



SYNDROME



WOLF-HIRSCHHORN
4p16.3
194190
WHSC1
6



SYNDROME







SUB TOTAL



52


Aneuploidy
chromosome1 associated
chromosome1


19



chromosome2 associated
chromosome2


20



chromosome3 associated
chromosome3


13



chromosome4 associated
chromosome4


15



chromosome5 associated
chromosome5


14



chromosome6 associated
chromosome6


14



chromosome7 associated
chromosome7


13



chromosome8 associated
chromosome8


15



chromosome9 associated
chromosome9


11



chromosome10 associated
chromosome10


13



chromosome11 associated
chromosome11


13



chromosome12 associated
chromosome12


12



chromosome13 associated
chromosome13


9



chromosome14 associated
chromosome14


10



chromosome15 associated
chromosome15


10



chromosome16 associated
chromosome16


9



chromosome17 associated
chromosome17


10



chromosome18 associated
chromosome18


10



chromosome19 associated
chromosome19


10



chromosome20 associated
chromosome20


10



chromosome21 associated
chromosome21


10



chromosome22 associated
chromosome22


10



chromosomeX associated
chromosomeX


17



chromosomeY associated
chromosomeY


11



SUB TOTAL



298


TOTAL




350









The clones whose localizations were identified by the above two methods, two end-sequencing and FISH, were used to correctly enumerate copy number of chromosome on which the clones localize. In this fashion, the probes for the entire human chromosomes can be pooled as a 24 set encompassing 1-22 plus X and Y sex chromosomes. In addition, even if the minimal affected regions (MAR) of microdeletions are much smaller, each of the 9 microdeletion syndrome regions is identified and the BAC clone DNAs to represent the region of a syndrome are pooled to detect the specific region of interest. The selected 350 BAC clones (probes) are summarized in Tables 2 to 33 as described above.


2.2. DNA Midi Prep


These 350 clones are extracted 72 clones per day using plasmid midi kit (Qiagen. 12145). After the extraction of DNA, the DNA is digested with Not-1 enzyme for 16 hours and run on a 1% agarose gel to check the purity and concentration. The DNAs are mixed according to chromosome number with same volume per clone. Total 24 kinds of DNA are prepared for the microarray chip.


2.3. Sonication & Condensation


To decrease the viscosity of high molecule DNA and to make the DNA size even about 3 Kbp, 1 ml of each pooled DNA was sonicated (Sonics & Materials, VCX750). After condensation, the pooled DNA was adjusted from 300 ng/μl to 400 ng/μl in 50% DMSO solution and put into 384 plate for spotting.


2.4. Pooling of BAC DNAs and Quality Control


For each pooled DNA to represent either a chromosome or a specific region of a chromosome (i.e., a region to represent a microdeletion syndrome), a list of BAC DNAs was used to combine equal volume of each BAC DNA into a properly labeled tube for a certain chromosome or specific region to be pooled (i.e., label chromosome 1 for chromosome 1 pooled BAC DNAs).


2.5. Confirmation of the Pooled BAC DNA by FISH


This pooled DNA is further validated before spotting by labeling the DNA for FISH validation. Subsequently, these pooled DNAs were used to validate the correct pooling of each chromosome or a region of chromosome by taking 1 ug of the DNA samples (pooled DNA and control) and labeling them with a green fluorescent dye, to perform FISH experiments as described above. The used pooled DAN and control samples were as shown in Table 35, and the regions used as controls were indicated in FIG. 3 in orange color.












TABLE 35







Target (pooled DNA)
Control









Chr.13
#626(13q12.1)



Chr.13
Cen.7



Chr.21
#88(21q11.2)



Chr.21
Cen.7



Chr.X
Cen.X










The obtained results were shown in FIGS. 4 to 8.


When each pooled DNA representing a chromosome is properly visualized in 10 independent metaphase spreads, the pooled probe was validated and used in the subsequent A-Chip fabrication. However, any of the pooled DNAs is not qualified, i.e., shows any contaminating chromosomal signals other than the projected chromosome (e.g., pooled 1 chromosome DNA probe binding and showing FISH signal from chromosome X and Y), this pooled DNA will be discarded and a new set of individual BAC DNA is prepared and pooled. This process was repeated until the proper signal was only obtained for the correct chromosome or a region within the chromosome.


2.6. Spotting


The microarray chip was manufactured by GeneMachine's OmniGrid100 (GeneMachines) using contact pin in controlled temperature of 22-25° C. and humidity of 50%. Each pooling clones were represented on an array as 5 times spots. The fabricated microarray chip was illustrated in FIG. 1. The specification of the microarray chip was summarized in Table 36. The fabricated microarray chip was named as MacArray™ A-Chip. The predecessor of the A-Chip, MacArray™ Karyo1400 utilizing the same technology has been approved by the Korean FDA in March of 2006 as an IVD test.









TABLE 36







5 times spotting


32 pooled clones


For 22 × 22 mm Coverglass & Maui DC Mixer










Coating
Amino-silanized
















Width
  25 ± 0.5 nm



Slide size
Length
  75 ± 0.5 nm




Thickness
  1 ± 0.1 nm



Spot
Spot area
8 × 7.5 nm (4 times)




Width space
0.64 nm




Length space
0.54 nm




Diameter
0.18 ± 0.05 nm



Subarray # in slide
Columns
2




Rows
2



Spot # in subarray
Columns
6




Rows
7










To attach the sonicated DNA on aminosilane coating surface (Corning, UltraGAPS), the microarray were baked in 80° C. for 2 hours and were applied 300 mJ of UV energy. The microarray was kept in desiccator until quality control or packaging.


The performance of used Omnigrid Spotter 100 (GeneMachines) was as follows:


Array run time: Using 48 pins on the optional server arm, 28,416 samples by deposited onto 100 slides in less than 10 hours.


Resolution: X and Y axis: 2.5 μm: Z axis: 1.25 μm


Repeatability: <+/−2.5 μm


Accurarcy: <+/−2.5 μm


2.7. Quality Control (Dye Stain & Hybridization Test)


There are 2 kinds of quality control method for MacArray™ A-Chip. One is dye staining for checking of spot morphology and concentration and the other is Hybridization test for checking the real performance in using normal test and reference genomic DNA. In this example, the Hybridization test was performed by GenePix 4000B (Axon). The GenePix 4000B (Axon) Specification was as follows:


Features 5 μm pixel size


dynamic monitoring of laser power


user-selectable laser power


user-selectable focus position


one-touch calibration and scanner matching


2.8: Prescan of Microarray


All produced microarrays were scanned in 532 nm, 10 micron resolution by the Axon Laser scanner 4000B and the results were saved as JPG images in the hard disk. It is easy to identify the spot shape. If a merged or scratched clones are on a microarray, the microarray are not passed.


2.9: Dye Staining


One slide per lot was stained with dye, like as Topro-3, ToTo-3 of Molecular Probes Inc. After dye staining, the microarray was scanned in 532 nm, 10 micron resolution by the Axon Laser scanner 4000B and the results were saved as JPG images in the hard disk. Clones which is not different from the background intensity was not above 1% in the whole clones.


2.10: Hybridization Test


At least 3 slides in the batches were tested with 10 types standard material genomic DNA and Klinefelter syndrome (karyotype: 47, XXY) genomic DNA. Table 37 shows the 10 types standard materials.












TABLE 37








Manufacturer*


No.
Syndrome
Karyotype
(Respiratory No.)


















1
Patau syndrome (Female)
47, XX, +13
Coriell (AG12070)


2
Patau syndrome (Male)
47, XY, +13
Coriell (GM00526)


3
Edward syndrome (Female)
47, XX, +18
Coriell (GM00143)


4
Edward syndrome (Male)
47, XY, +18
Coriell (GM01359)


5
Down syndrome (Female)
47, XX, +21
Coriell (GM03606)


6
Down syndrome (Male)
47, XY, +21
Coriell (AG05397)


7
Turner syndrome
45, XO
Coriell (GM10179)


8
Klinefelter syndrome
47, XXY
Coriell (GM03091)


9
Normal Female
46, XX
Coriell (GM08400)


10
Normal Male
46, XY
Coriell (GM08402)





*Manufacturer: Coriell Institute for Medical Research, USA






After hybridization test, each sample log 2T/R ratio mean value have to satisfy the criteria values as shown in Table 38. Table 38 shows the criteria values of 10 type standard materials











TABLE 38









Chromosome log2 T/R Ratio Mean Value











Target
Chromosome 1~22












chrom
Except
Sex chromosome














No.
Syndrome
Karyotype
osome
Target
target
X
Y

















1
Patau (Female)
47, XX, +13
13
M ≧ 0.250
−0.250 <
−0.250 <
M ≦ −0.400







M < 0.250
M < 0.250


2
Patau (Male)
47, XY, +13

M ≧ 0.250
−0.250 <
M ≦ −0.250
−0.400 <







M < 0.250

M < 0.400


3
Edward (Female)
47, XX, +18
18
M ≧ 0.250
−0.250 <
−0.250 <
M ≦ −0.400







M < 0.250
M < 0.250


4
Edward (Male)
47, XY, +18

M ≧ 0.250
−0.250 <
M ≦ −0.250
−0.400 <







M < 0.250

M < 0.400


5
Down (Female)
47, XX, +21
21
M ≧ 0.250
−0.250 <
−0.250 <
M ≦ −0.400







M < 0.250
M < 0.250


6
Down (Male)
47, XY, +21

M ≧ 0.250
−0.250 <
M ≦ −0.250
−0.400 <







M < 0.250

M < 0.400


7
Turner
45, XO
X, Y


M ≦ −0.250
M ≦ −0.400


8
Klinefelter
47, XXY
X, Y


−0.250 <
−0.400 <








M < 0.250
M < 0.400


9
Normal (Female)
46, XX


−0.250 <
−0.250 <
M ≦ −0.400







M < 0.250
M < 0.250


10
Normal (Male)
46, XY


−0.250 <
M ≦ −0.250
−0.400 <







M < 0.250

M < 0.400





* M: Mean value of chromosomal log2 T/R(Test/Reference) signal ratio






Also the average of log2T/R ratio SD (Standard Deviation) value from Chromosome 1 to Chromosome 22 clones except target chromosomes (e.g., chromosome 13, 18 or 21) has to be below than 0.08.


Example 3
Sample/Specimen Collection

3.1: Pre-Processing of Samples


Before Starting DNA Extraction Pre-Processing


Set a heat block to 55° C.


Reagents: Prepare RBC Lysis Solution (1), Cell Lysis Solution (2), Proteinase K (3).


Reagents 1 and 2 were treated at room temperature, reagent 3 was spun down and put on ice.


Amniotic Fluid Pre-Processing

    • {circle around (1)} 4 mL of amniotic fluid was put into a 15 mL conical tube and centrifuge at 1,500 rpm for 5 minutes at room temperature (RT).
    • {circle around (2)} The top layer 3.5 mL was pipetted out and the bottom layer was vortexed for 10 seconds.
    • {circle around (3)} 0.5 mL vortexed solution was transferred into a 1.5 mL eppendorf tube and centrifuged at 13,000 rpm for 1 minute at RT.
    • {circle around (4)} The supernatant was removed and while leaving just a cell pellet.
    • {circle around (5)} 600 μL Cell Lysis Solution was added and vortexed for 5 seconds.
    • {circle around (6)} The tube into was put a heat block at 55° C. 15 minutes.
    • {circle around (7)} The obtained reaction mixture was cooled down at RT for 5 minutes before extracting DNA.


Chorionic Villi Pre-Processing

    • {circle around (1)} 5 mL PBS was put into two petri dishes.
    • {circle around (2)} Blood was removed from the sample by washing consecutively in the two petri dishes.
    • {circle around (3)} The sample was put into a new petri dish and dissected out the decidua under a dissecting microscope.
    • {circle around (4)} The sample was transferred into a new petri dish and diced into 2 mm blocks.
    • {circle around (5)} The diced samples were put into a 1.5 mL eppendorf tube and 600 μL of Cell Lysis Solution, and 84 of Proteinase K were added thereto.
    • {circle around (6)} The obtained reaction mixture was put into a 55° C. heat block for two hours and vortexed for 5 seconds in every 30 minutes.
    • {circle around (7)} The obtained reaction mixture was cooled down at RT for 5 minutes before extracting DNA.


Cord Blood and Peripheral Blood Pre-Processing

    • {circle around (1)} 300 μL of blood was put into a 1.5 mL eppendorf tube, 900 μL of RBC Lysis Solution was added, and vortexed for 10 seconds.
    • {circle around (2)} The reaction mixture was stored at RT for 5 minutes and spun at 13,000 rpm for 1 minute.
    • {circle around (3)} The supernatant was removed and while leaving just a cell pellet.
    • {circle around (4)} 600 μL Cell Lysis Solution was added and vortex for 5 seconds.
    • {circle around (5)} The tube was put into a heat block at 55° C. 15 minutes.
    • {circle around (6)} The reaction mixture was cooled down at RT for 5 minutes before extracting DNA.


Placenta Villi Pre-Processing

    • {circle around (1)} 5 mL PBS was put into two petri dishes.
    • {circle around (2)} Blood was removed from the sample by washing consecutively in the two petri dishes.
    • {circle around (3)} The sample was transferred into a new petri dish and diced into 2 mm blocks.
    • {circle around (4)} The diced samples were put into a 1.5 mL eppendorf tube and put 600 μL of Cell Lysis Solution, 8 μL of Proteinase K.
    • {circle around (5)} The reaction mixture was put into a 55° C. heat block for two hours and vortex for 5 seconds in every 30 minutes.
    • {circle around (6)} The reaction mixture was cooled down at RT for 5 minutes before extracting DNA.


Tissue Culture Cells Pre-Processing

    • {circle around (1)}15 μL (3×106 Cells) of culture cells was put into a 1.5 mL eppendorf tube and 600 μL of Cell Lysis Solution and 8 μL of Proteinase K were added thereto.
    • {circle around (2)} The reaction mixture was put into a 55° C. heat block for two hours and vortex for 5 seconds in every 30 minutes.
    • {circle around (3)} The reaction mixture was cooled down at RT for 5 minutes before extracting DNA.


3.2. DNA Extraction Processing Protocols


Before Starting DNA Extraction Processing


Set a heat block to 55° C.


Reagents: Glycogen solution (4), RNase A (5), Protein Precipitation Solution (6), DNA Hydration Solution (7)


Spin down reagents 4, 5, 6, & 7 and store at RT.


Protocol

    • {circle around (1)} Add 2 μL of RNase A into the pre-processed sample containing tube and vortex for 10 seconds.
    • {circle around (2)} Digest for 15 minutes at a 37° C. incubator.
    • {circle around (3)} Add 150 μL of Protein Precipitation Solution.
    • {circle around (4)} Vortex for 10 seconds and store on ice for 5 minutes.
    • {circle around (5)} Centrifuge for 4 minutes at 13,000 rpm at RT and take about 700 μL of the supernatant into a new 1.5 mL eppendorf tube.
    • {circle around (6)} Add 600 μL of Isopropanol and 1 μL Glycogen solution and votex for 3 seconds.
    • {circle around (7)} Remove the supernatant by spinning at 13,000 rpm for 10 minutes at RT and add 150 uL of 70% ethanol.
    • {circle around (8)} Spin at 13,000 rpm for 5 minutes at RT and remove the supernatant.
    • {circle around (9)} Dry at RT for 10 minutes.
  • {circle around (10)} Add 1004 of DNA Hydration Solution (except for the DNA from amniotic fluids, put 25 μL of DNA Hydration Solution).
    • {circle around (11)} Set a heat block to 55° C.
    • {circle around (12)} Vortex for 3 seconds and store at −20° C.


3.3. Quality Control of Extracted DNA


Before Starting the DNA Quality Check


2×DNA Loading Dye Preparation: Mix sterile 200 μL ddH2O and 100 μ 6×DNA Loading Dye in a 1.5 mL eppendorf tube and votex for 3 seconds and spin down briefly.


50 ng/μl λHindIII Marker Preparation: Mix 50 μL of 6×DNA Loading Dye and 30 μL of 500 ng/μL λHindIII Marker in 250 μL sterile ddH2O in a 1.5 mL.


(1) Agarose Gel Electrophoresis

    • {circle around (1)} Agarose powder was melted by microwaving in 1×TAE Buffer to make 1% Agarose gel.
      • A. DNA extracted from Blood (Cord and Periperal blood), Tissue (Corionic and Placental villi) and tissue culture cells: Mix, in a 1.5 mL eppendorf tube, 10 μL extracted DNA and 90 μL sterile ddH2O, then vortex for 3 seconds and spin down. 4 μL of this diluted DNA solution is mixed with 4 μL of 2×DNA Loading Dye.
      • B. DNA extracted from amniotic fluid: Mix 4 μL of DNA straight with 4 μL of 2×DNA Loading Dye.
    • {circle around (2)} 1% Agarose gel was put into a gel chamber with 1×TAE Buffer.
    • {circle around (3)} 4 μL of 50 ng/μL λHindIII Marker, 8 μL of 2×DNA Loading Dye plus DNA was loaded on a separate well.
    • {circle around (4)} The 2×DNA Loading Dye 30 mm was run and the gel electrophoris was completed.
    • {circle around (5)} The 1% Agarose gel was converted into a Gel image analyzer and some images were captured.


(2) λHindIII Marker Separation in 1% Agarose Gel


The obtained results were summarized in Table 39 and FIG. 9.











TABLE 39





Band No.
Concentration(approx.)
Size (kb)

















7
25.0 ng/μl 
23.0


6
10.0 ng/μl 
9.4


5
7.0 ng/μl
6.5









4
N/A










3
2.5 ng/μl
2.3


2
2.2 ng/μl
2.0


1
0.5 ng/μl
0.5









(3) Measure DNA Concentration Using UV/Spectrophotometric Detector.


The measurement was performed according to the manufacturer's protocol, but briefly, the extracted DNA was diluted in ddH2O and Optical Density (OD) was measured at 260 and 280 nm wavelength.


Example 3
DNA Detection

The following detection processes were schematically shown in FIG. 10.


3.1. Prepare Genomic DNA from the Samples


The sample DNA was collected in the amount of 50 ng from the amniotic sample, and 500 ng from the rest of test samples.


The used test samples were summarized in Tables 40 and 41.












TABLE 40








Manufacturer*


No.
Syndrome
Karyotype
(Repository No.)


















1
Patau syndrome (Male)
47, XY, +13
Coriell (NA02948)


2
Edward syndrome (Male)
47, XY, +18
Coriell (NA01359)


3
Edward syndrome
47, XX, +18
Coriell (GM00143)



(Female)


4
Down syndrome (Male)
47, XY, +21
Coriell (NA01921)


5
Turner syndrome
45, XO
Coriell (GM00857)


6
Klinefelter syndrome
47, XXY
Coriell (GM03091)


7
Super female syndrome
47, XXX
Coriell (NA04626)


8
Super male syndrome
47, XYY
Coriell (GM02587)


9
Normal Female
46, XX
Coriell (GM08400)


10
Normal Male
46, XY
Coriell (GM08402)


11
Wolf-Hirschhorn
4p16.3 deletion
Coriell (GM00343)



syndrome


12
Cri-du-chat syndrome
5p15.2 deletion
Coriell (NA14124)


13
Prader-Willi syndrome
15q11-q13
Coriell (NA11382)




deletion


14
Smith-Magenis syndrome
17p11.2 deletion
Coriell (GM13476)


15
Digeorge syndrome
22q11.2 deletion
Coriell (NA07215)


16
Steroid sulfatase
Xp22.31 deletion
Coriell (GM03035)



deficiency disease





















TABLE 41








Number
Number of
Number





of
Correct
of Mis-





Samples
Karyo-
karyo-


No
Sample
Karyotype
tested
typing
typing




















1
Patau syndrome
47, XY, +13
8
8
0



(Male)


2
Edward syndrome
47, XY, +18
8
8
0



(Male)


3
Edward syndrome
47, XX, +18
7
7
0



(Female)


4
Down syndrome
47, XY, +21
5
5
0



(Male)


5
Turner syndrome
45, XO
8
8
0


6
Klinefelter
47, XXY
8
8
0



syndrome


7
Super female
47, XXX
8
8
0



syndrome


8
Super male
47, XYY
8
8
0



syndrome


9
Normal Female
46, XX
8
8
0


10
Normal Male
46, XY
8
8
0


11
Wolf-Hirschhorn
4p16.3
6
6
0



syndrome
deletion


12
Cri-du-chat
5p15.2
9
9
0



syndrome
deletion


13
Prader-Willi
15q11-q13
9
9
0



syndrome
deletion


14
Smith-Magenis
17p11.2
7
7
0



syndrome
deletion


15
Digeorge
22q11.2
8
8
0



syndrome
deletion


16
Steroid sulfatase
Xp22.31
7
7
0



deficiency
deletion






total

122
122
0









3.2. DNA Labeling with Fluorescent Dyes


Sample DNA 50 ng or 500 ng+1 mM Cy3 3 μL


Reference DNA 50 ng or 500 ng+1 mM Cy5 3 μL


Temperature: 37° C.


Reaction Time: 16 hours (or overnight)


After reaction labeled DNA amount was equaled or more than 4 μg.


3.3. Hybridization


Cy3-Sample DNA and Cy5-Reference DNA were mixed. The obtained mixture was applied onto the Macrogen A-Chip fabricated above and then covered with a coverglass, to allow hybridization.


Temperature: 37±1° C., humidity 90±5%


Reaction time: 16 hours (or Overnight)


3.4. Washing


Wash 1: 50% Formamide, 2×SSC, 46° C., 15 minutes (min.)


Wash 2: 2×SSC, 0.1% SDS, 46° C., 30 min.


Wash 3: 1×Phosphate Buffer, 0.1% Nonidet P40, RT, 15 min.


Wash 4: 2×SSC, RT, 5 min.


Wash 5: 70%, 85%, 100% ethanol, RT


Dry Spin: 1,500 rpm, RT, 5 min.


3.5. Scanning & Image Analysis


Refer to cutoff table for scanning time.


Refer to MacArray A-Chip manual for image analysis protocols


The obtained results were shown in Tables 42 and 43, and FIGS. 11A and 11B.














TABLE 42










chr1~chr22





Target
(except target)
chrX
chrY





















STD

STD

STD

STD


No
Sample
Karyotype
AVG
EV
AVG
EV
AVG
EV
AVG
EV




















1
Patau (Male)
47, XY, +13
0.398
0.032
0.001
0.034






2
Edward (Male)
47, XY, +18
0.461
0.052
0.001
0.040






3
Edward (Female)
47, XX, +18
0.420
0.039
0.000
0.037






4
Down (Male)
47, XY, +21
0.290
0.027
0.004
0.043






5
Turner
45, XO


0.004
0.040
−0.449
0.102
−0.574
0.201


6
Klinefelter
47, XXY


0.004
0.034
−0.024
0.015
−0.003
0.015


7
Super female
47, XXX


0.004
0.040
0.336
0.053
−0.488
0.174


8
Super male
47, XYY


−0.001
0.046
−0.473
0.087
0.360
0.071


9
Normal Female
46, XX


0.004
0.031
0.004
0.027
−0.556
0.082


10
Normal Male
46, XY


0.001
0.036
−0.516
0.065
−0.017
0.041


11
Wolf-Hirschhorn
4p16.3 del
−0.447
0.123
0.001
0.036






12
Cri-du-chat
5p15.2 del
−0.495
0.110
−0.003
0.047






13
Prader-Willi
15q11-q13 del
−0.525
0.062
0.001
0.037






14
Smith-Magenis
17p11.2 del
−0.390
0.063
−0.001
0.033






15
Digeorge
22q11.2 del
−0.330
0.052
0.004
0.037






16
Steroid sulfatase
Xp22.31 del
−1.145
0.179
0.001
0.040







deficiency


















TABLE 43









Chromosome log2 T/R Ratio Mean Value











Target
Chromosome 1~22












chromo-
Except
Sex chromosome














No
Syndrome
Karyotype
some
Target
target
X
Y

















1
Patau (Male)
47, XY, +13
13
M ≧ 0.250
−0.250 <
M ≦ −0.250
−0.400 <







M < 0.250

M < 0.400


2
Edward (Male)
47, XY, +18
18
M ≧ 0.250
−0.250 <
M ≦ −0.250
−0.400 <







M < 0.250

M < 0.400


3
Edward (Female)
47, XX, +18

M ≧ 0.250
−0.250 <
−0.250 <
M ≦ −0.400







M < 0.250
M < 0.250


4
Down (Male)
47, XY, +21

M ≧ 0.250
−0.250 <
M ≦ −0.250
−0.400 <







M < 0.250

M < 0.400


5
Turner
45, XO
X, Y


M ≦ −0.250
M ≦ −0.400


6
Klinefelter
47, XXY
X, Y


−0.250 <
−0.400 <








M < 0.250
M < 0.400


7
Super Female
47, XXX
X, Y


M ≦ −0.250
M ≦ −0.400


8
Super Male
47, XYY
X, Y


−0.250 <
−0.400 <








M < 0.250
M < 0.400


9
Normal (Female)
46, XX


−0.250 <
−0.250 <
M ≦ −0.400







M < 0.250
M < 0.250


10
Normal (Male)
46, XY


−0.250 <
M ≦ −0.250
−0.400 <







M < 0.250

M < 0.400


11
Wolf-Hirschhorn
4p16.3 del
4p16.3
M ≦ −0.250
−0.250 <









M < 0.250


12
Cri-du-chat
5p15.2 del
5p15.2
M ≦ −0.250
−0.250 <









M < 0.250


13
Prader-Willi
15q11-q13 del
15q11-q13
M ≦ −0.250
−0.250 <









M < 0.250


14
Smith-Magenis
17p11.2 del
17p11.2
M ≦ −0.250
−0.250 <









M < 0.250


15
Digeorge
22q11.2 del
22q11.2
M ≦ −0.250-
−0.250 <









M < 0.250


16
Steroid sulfatase
Xp22.31 del
Xp22.31
M ≦ −0.800-
−0.250 <





deficiency



M < 0.250








Claims
  • 1. A microarray chip for detecting human chromosomal abnormality, comprising one or more pooled probe sets immobilized in spots on a substrate, wherein the pooled probe set is one or more selected from the group consisting of: a pooled probe set (pooled probe set 1) specific to the chromosomal abnormality in copy number of Chromosome 1 consisting essentially of human chromosomal polynucleotides carried in BAC27_N16, BAC25_C19, BAC153_I07, BAC217_C19, BAC59_D13, BAC54_I02, BAC163_C09, BAC218_G03, BAC152_F22, BAC34_P03, BAC36_I16, BAC145_L11, BAC37_O23, BAC239_G19, BAC105_P13, BAC57_N17, BAC239_A12, BAC171_H09, and BAC222_E02;a pooled probe set (pooled probe set 2) specific to the chromosomal abnormality in copy number of Chromosome 2 consisting essentially of human chromosomal polynucleotides carried in BAC126_E04, BAC197_E10, BAC43_A02, BAC33_C05, BAC59_D21, BAC12_G01, BAC141_F07, BAC163_C22, BAC36_H22, BAC143_G24, BAC238_G01, BAC252_A16, BAC46_J12, BAC57_C12, BAC34_F17, BAC79_L21, BAC39_M07, BAC156_K09, BAC195_I06, and BAC88_K20;a pooled probe set (pooled probe set 3) specific to the chromosomal abnormality in copy number of Chromosome 3 consisting essentially of human chromosomal polynucleotides carried in BAC197_B21, BAC158_C03, BAC144_C11, BAC186_N05, BAC103_F06, BAC114_B23, BAC102_E23, BAC119_G21, BAC68_H20, BAC237_M11, BAC168_G04, BAC61_M02, and BAC36_N19;a pooled probe set (pooled probe set 4) specific to the chromosomal abnormality in copy number of Chromosome 4 consisting essentially of human chromosomal polynucleotides carried in BAC60_H08, BAC26_C10, BAC68—O19, BAC102_G08, BAC127_B16, BAC176_G14, BAC41—O05, BAC37_H04, BAC115_C13, BAC30_N21, BAC220_D24, BAC106_P17, BAC41_O11, BAC157_P10, and BAC27_L15;a pooled probe set (pooled probe set 5) specific to the chromosomal abnormality in copy number of Chromosome 5 consisting essentially of human chromosomal polynucleotides carried in BAC86_B20, BAC33_N18, BAC55_L24, BAC226_H03, BAC156_E24, BAC237_B02, BAC29_D17, BAC139_M23, BAC21_J16, BAC27_N23, BAC148_D23, BAC186_L21, BAC238_E21, and BAC175_N07;a pooled probe set (pooled probe set 6) specific to the chromosomal abnormality in copy number of Chromosome 6 consisting essentially of human chromosomal polynucleotides carried in BAC125_G09, BAC182_E20, BAC81_C08, BAC24_P12, BAC76_A23, BAC26_F16, BAC43_M14, BAC27_P17, BAC1_N23, BAC247_D17, BAC101_M04, BAC90_F08, BAC118_M18, and BAC179_N12;a pooled probe set (pooled probe set 7) specific to the chromosomal abnormality in copy number of Chromosome 7 consisting essentially of human chromosomal polynucleotides carried in BAC231_L03, BAC82_L17, BAC218_N01, BAC5_A09, BAC170_M16, BAC119_K16, BAC248_P06, BAC96_F02, BAC139_J04, BAC76_K13, BAC192_N04, BAC154_A21, and BAC120_I09;a pooled probe set (pooled probe set 8) specific to the chromosomal abnormality in copy number of Chromosome 8 consisting essentially of human chromosomal polynucleotides carried in BAC150_M15, BAC149_J08, BAC63_M21, BAC147_O15, BAC44_I16, BAC30_N24, BAC43_J01, BAC234_M17, BAC68_K11, BAC200_C08, BAC237_M08, BAC61_N10, BAC80_H19, BAC150_P12, and BAC66_I02;a pooled probe set (pooled probe set 9) specific to the chromosomal abnormality in copy number of Chromosome 9 consisting essentially of human chromosomal polynucleotides carried in BAC80_F23, BAC28_L14, BAC137_L16, BAC161_C10, BAC92_D01, BAC163_H11, BAC12_E22, BAC172_D10, BAC149_L08, BAC188_O18, and BAC126_N07;a pooled probe set (pooled probe set 10) specific to the chromosomal abnormality in copy number of Chromosome 10 consisting essentially of human chromosomal polynucleotides carried in BAC170_F05, BAC102_J19, BAC40_P04, BAC141_E23, BAC246_I22, BAC14_K16, BAC52_B14, BAC158_C10, BAC155_O18, BAC144_E19, BAC218_E11, BAC48_I12, and BAC182_N07;a pooled probe set (pooled probe set 11) specific to the chromosomal abnormality in copy number of Chromosome 11 consisting essentially of human chromosomal polynucleotides carried in BAC68_K10, BAC90_E18, BAC24_K17, BAC58_O19, BAC36_K05, BAC150_P20, BAC154_H22, BAC26_C09, BAC119_O13, BAC195_O14, BAC73_E17, BAC 142_K09, and BAC65_D19;a pooled probe set (pooled probe set 12) specific to the chromosomal abnormality in copy number of Chromosome 12 consisting essentially of human chromosomal polynucleotides carried in BAC60_I23, BAC121_P21, BAC199_G02, BAC65_G10, BAC41_I18, BAC10_M07, BAC39_O14, BAC144_K11, BAC178_M15, BAC134_M17, BAC65_I21, and BAC27_E08;a pooled probe set specific (pooled probe set 13) to the chromosomal abnormality in copy number of Chromosome 13 consisting essentially of human chromosomal polynucleotides carried in BAC28_H21, BAC163_F01, BAC78_C21, BAC135_O03, BAC237_P24, BAC84_N09, BAC8_C18, BAC133_G23, and BAC116_B15;a pooled probe set (pooled probe set 14) specific to the chromosomal abnormality in copy number of Chromosome 14 consisting essentially of human chromosomal polynucleotides carried in BAC236_F24, BAC22_E01, BAC37_K09, BAC79_J20, BAC50_I09, BAC15_E12, BAC63_O11, BAC11_N10, BAC39_P02, and BAC101_O15;a pooled probe set (pooled probe set 15) specific to the chromosomal abnormality in copy number of Chromosome 15 consisting essentially of human chromosomal polynucleotides carried in BAC66_K21, BAC162_K11, BAC178_K16, BAC21_K13, BAC167_M02, BAC88_F18, BAC168_F12, BAC10_E08, BAC177_H09, and BAC41_K03;a pooled probe set (pooled probe set 16) specific to the chromosomal abnormality in copy number of Chromosome 16 consisting essentially of human chromosomal polynucleotides carried in BAC38_I04, BAC96_J19, BAC120_K24, BAC177_P23, BAC247_B03, BAC117_H14, BAC96_G02, BAC24_D17, and BAC223_D19;a pooled probe set (pooled probe set 17) specific to the chromosomal abnormality in copy number of Chromosome 17 consisting essentially of human chromosomal polynucleotides carried in BAC200_M05, BAC50_A03, BAC149_H11, BAC29_G13, BAC238_E06, BAC150_O15, BAC70_P11, BAC70_N11, BAC116_E10, and BAC48_K14;a pooled probe set (pooled probe set 18) specific to the chromosomal abnormality in copy number of Chromosome 18 consisting essentially of human chromosomal polynucleotides carried in BAC57_H08, BAC141_I04, BAC252_H16, BAC232_E19, BAC149_I18, BAC186_P19, BAC 151_L02, BAC230_C11, BAC43_A24, and BAC184_J04;a pooled probe set (pooled probe set 19) specific to the chromosomal abnormality in copy number of Chromosome 19 consisting essentially of human chromosomal polynucleotides carried in BAC178_L22, BAC160_C11, BAC131_N13, BAC54_N22, BAC233_K14, BAC162_K04, BAC76_E22, BAC211_B15, BAC101_H02, and BAC193_C07;a pooled probe set (pooled probe set 20) specific to the chromosomal abnormality in copy number of Chromosome 20 consisting essentially of human chromosomal polynucleotides carried in BAC247_K09, BAC26_J24, BAC75_H16, BAC37_M13, BAC19_G17, BAC82_B07, BAC96_H08, BAC166_J02, BAC41_E11, and BAC146_N07;a pooled probe set (pooled probe set 21) specific to the chromosomal abnormality in copy number of Chromosome 21 consisting essentially of human chromosomal polynucleotides carried in BAC102_F10, BAC240_M07, BAC200_O02, BAC97_O19, BAC119_K07, BAC200_A23, BAC221_D22, BAC100_D11, BAC33_D15, and BAC126_M10;a pooled probe set (pooled probe set 22) specific to the chromosomal abnormality in copy number of Chromosome 22 consisting essentially of human chromosomal polynucleotides carried in BAC169_G07, BAC153_I19, BAC100_P10, BAC37_J03, BAC187_K08, BAC131_H09, BAC106_C07, BAC66_M06, BAC51_M21, and BAC153_O04;a pooled probe set (pooled probe set 23) specific to the chromosomal abnormality in copy number of Chromosome X consisting essentially of human chromosomal polynucleotides carried in BAC70_N16, BAC22_H14, BAC65_L14, BAC151_A03, BAC49_G05, BAC130_K20, BAC103_N15, BAC136_M01, BAC6_B17, BAC141_P03, BAC246_K02, BAC91_J24, BAC97_C11, BAC63_G23, BAC73_B07, BAC162_B10, and BAC119_C15;a pooled probe set (pooled probe set 24) specific to the chromosomal abnormality in copy number of Chromosome Y consisting essentially of human chromosomal polynucleotides carried in BAC24_K23, BAC205_L13, BAC127_H21, BAC192_M14, BAC101_I21, BAC140_H17, BAC65_J16, BAC180_K16, BAC102_F03, BAC31_L01, and BAC240_H05;a pooled probe set (pooled probe set 25) specific to micro-deletion of 4p16.3 of Chromosome 4 consisting essentially of human chromosomal polynucleotides carried in BAC50_H08, BAC67_I12, BAC100_E03, BAC1_F06, BAC135_O20, and BAC153_J14;a pooled probe set (pooled probe set 26) specific to micro-deletion of 5p15.2 of Chromosome 5 consisting essentially of human chromosomal polynucleotides carried in BAC143_N22, BAC206_I13, BAC252_N08, BAC64_P22, BAC208_N21, BAC200_E05, and BAC240_K06;a pooled probe set (pooled probe set 27) specific to micro-deletion of 7q11.2 of Chromosome 7 consisting essentially of human chromosomal polynucleotides carried in BAC69_O08, BAC66_N22, BAC180_N24, BAC67_C05, BAC183_A12, and BAC123_D05;a pooled probe set (pooled probe set 28) specific to micro-deletion of 15q11-15q13 of Chromosome 15 consisting essentially of human chromosomal polynucleotides carried in BAC188_N24, BAC223_H02, BAC217_F02, BAC71_A18, BAC5_L18, BAC248_C13, BAC78_F07, BAC180_J22, BAC21_O06, and BAC105_L07;a pooled probe set (pooled probe set 29) specific to micro-deletion of 17p13.3 of Chromosome 17 consisting essentially of human chromosomal polynucleotides carried in BAC95_J10, BAC75_C17, BAC110_O13, BAC63_J08, BAC190_F10, BAC186_M15, BAC183_M06, BAC135_N07, BAC148_F06, and BAC31_H03;a pooled probe set (pooled probe set 30) specific to micro-deletion of 17p11.2 of Chromosome 17 consisting essentially of human chromosomal polynucleotides carried in BAC249_G12, BAC41_D18, and BAC186_E14;a pooled probe set (pooled probe set 31) specific to micro-deletion of 22q11.2 of Chromosome 22 consisting essentially of human chromosomal polynucleotides carried in BAC124_E21, BAC196_A22, BAC69_P21, BAC141_K20, BAC169_K21, BAC145_P12, and BAC224_F10; anda pooled probe set (pooled probe set 32) specific to micro-deletion of Xp22.31 of Chromosome X consisting essentially of human chromosomal polynucleotides carried in BAC221_A12, BAC191_E24, and BAC231_F19.
  • 2. The microarray chip according to claim 1, comprising one or more selected form the group consisting of pooled probe sets 1 to 24 to detect chromosomal abnormality in copy number of Chromosomes 1 to 22, X and Y corresponding to the used pooled probe set.
  • 3. The microarray chip according to claim 1, comprising one or more selected form the group consisting of pooled probe sets 25 to 32 to detect micro-deletion of specific chromosomal regions corresponding to the used pooled probe set.
  • 4. The microarray chip according to claim 2, further comprising one or more selected form the group consisting of pooled probe sets 25 to 32, to simultaneously detect chromosomal abnormality in copy number of Chromosomes 1 to 22, X and Y, and micro-deletion of specific chromosomal regions corresponding to the used pooled probe sets.
  • 5. The microarray chip according to claim 1, comprising all pooled probe sets 1 to 32, to simultaneously detect chromosomal abnormality in copy number of Chromosomes 1 to 22, X and Y, and micro-deletion of specific chromosomal regions.
  • 6. A method of preparing the microarray chip according to claim 1, comprising the step of immobilizing one or more selected from the group consisting of pooled probe sets 1 to 32 in spots on a substrate, wherein all probes belonging to only a pooled probe set are immobilized in one spot, one spot comprises only one pooled probe set, and the microarray chip comprises at least one spot for each pooled probe set.
  • 7. A method of detecting chromosomal abnormalities, comprising the steps of: providing the microarray chip according to claim 1;labeling a test sample DNA and a reference DNA with different labels from each other;fragmentizing the labeled DNAs, and applying the obtained test sample and reference DNA fragments onto the spots on the microarray chip, respectively, to hybridize the DNA fragments with the probes in the spot;measuring a signal intensity from each pooled probe set hybridized with the test sample DNA or the reference DNA; andcomparing the signal intensity from the test sample DNA over that from the reference DNA.
  • 8. The method according to claim 7, wherein the microarray chip comprises one or more selected form the group consisting of pooled probe sets 1 to 24, to detect chromosomal abnormality in copy number of Chromosomes 1 to 22, X and Y corresponding to the used pooled probe set.
  • 9. The method according to claim 7, wherein the microarray chip comprises one or more selected form the group consisting of pooled probe sets 25 to 32 to detect micro-deletion of specific chromosomal regions corresponding to the used pooled probe set.
  • 10. The method according to claim 9, wherein the microarray chip further comprises one or more selected form the group consisting of pooled probe sets 25 to 32, to simultaneously detect chromosomal abnormality in copy number of Chromosomes 1 to 22, X and Y, and micro-deletion of specific chromosomal regions corresponding to the used pooled probe sets.
  • 11. The method according to claim 7, wherein the microarray chip comprises all pooled probe sets I to 32, to simultaneously detect chromosomal abnormality in copy number of Chromosomes 1 to 22, X and Y, and micro-deletion of specific chromosomal regions.
  • 12. The method according to claim 7, wherein the test sample DNA was extracted from amniotic fluid, peripheral blood, chorionic villus, umbilical cord blood, placenta villi, and cultured cells obtained from human.
  • 13. A kit for diagnosing a disease associated with a chromosomal abnormality, comprising the microarray chip according to claim 1.
  • 14. The kit according to claim 13, wherein the microarray chip comprises one or more selected from the group consisting of: pooled probe set 13 for diagnosing Patau syndrome;pooled probe set 18 for diagnosing Edward syndrome;pooled probe set 21 for diagnosing Down syndrome;pooled probe sets 23 and 24 for diagnosing Tuner syndrome, Klinefelter syndrome, Super female syndrome, or Super male syndrome;pooled probe set 25 for diagnosing Wolf-Hirschhorn syndrome;pooled probe set 26 for diagnosing Cri-Du-Chat syndrome;pooled probe set 27 for diagnosing William syndrome;pooled probe set 28 for diagnosing Prader-willi syndrome or Angelman syndrome;pooled probe set 29 for diagnosing Miller-Dieker Lissencephaly syndrome;pooled probe set 30 for diagnosing Smith-Magenis syndrome;pooled probe set 31 for diagnosing Digeorge syndrome; andpooled probe set 32 for diagnosing Steroid Sulfatase deficiency syndrome.
  • 15. The kit according to claim 14, wherein the microarray chip comprises of pooled probe sets 13, 18, 21, and 23 to 32 for simultaneously diagnosing Down syndrome, Patau syndrome, Edward syndrome, Tuner syndrome, Klinefelter syndrome, Super female syndrome, Super male syndrome, Wolf-Hirschhorn syndrome, Cri-Du-Chat syndrome, William syndrome, Prader-willi syndrome, Angelman syndrome, Miller-Dieker Lissencephaly syndrome, Smith-Magenis syndrome, Digeorge syndrome, and Steroid Sulfatase deficiency syndrome.
  • 16. A method of diagnosing a disease associated with a chromosomal abnormality, comprising the steps of providing a microarray chip according to claim 1;labeling a test sample DNA from a patient and a reference DNA with different labels from each other;fragmentizing the labeled DNAs, and applying the obtained test sample and reference DNA fragments onto the spots on the microarray chip, respectively, to hybridize the DNA fragments with the probes;measuring a signal intensity from each probe hybridized with the test sample DNA or the reference DNA; andcomparing the signal intensity from the test sample DNA over that from the reference DNA, to determine that the patient has a disease associated with a chromosomal abnormality detectable by the used pooled probe set, when a difference is detected between the signal intensity from the test sample DNA and that from the reference DNA.
  • 17. The method according to claim 16, wherein the microarray chip comprises one or more selected from the group consisting of: pooled probe set 13 for diagnosing Patau syndrome;pooled probe set 18 for diagnosing Edward syndrome;pooled probe set 21 for diagnosing Down syndrome;pooled probe sets 23 and 24 for diagnosing Tuner syndrome, Klinefelter syndrome, Super female syndrome, or Super male syndrome;pooled probe set 25 for diagnosing Wolf-Hirschhorn syndrome;pooled probe set 26 for diagnosing Cri-Du-Chat syndrome;pooled probe set 27 for diagnosing William syndrome;pooled probe set 28 for diagnosing Prader-willi syndromev or Angelamn syndrome;pooled probe set 29 for diagnosing Miller-Dieker Lissencephaly syndrome;pooled probe set 30 for diagnosing Smith-Magenis syndrome;pooled probe set 31 for diagnosing Digeorge syndrome; andpooled probe set 32 for diagnosing Steroid Sulfatase deficiency syndrome.
  • 18. The method according to claim 14, wherein the microarray chip comprises of pooled probe sets 13, 18, 21, and 23 to 32, to simultaneously diagnose Down syndrome, Patau syndrome, Edward syndrome, Tuner syndrome, Klinefelter syndrome, Super female syndrome, Super male syndrome, Wolf-Hirschhorn syndrome, Cri-Du-Chat syndrome, William syndrome, Prader-willi syndrome, Angelman syndrome, Miller-Dieker Lissencephaly syndrome, Smith-Magenis syndrome, Digeorge syndrome, and Steroid Sulfatase deficiency syndrome.
Priority Claims (1)
Number Date Country Kind
60843372 Sep 2006 US national
CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of U.S. Provisional Application No. 60/843,372 filed on Sep. 8, 2006, which is hereby incorporated by reference for all purposes as if fully set forth herein.

PCT Information
Filing Document Filing Date Country Kind 371c Date
PCT/KR2007/004345 9/7/2007 WO 00 4/20/2010