KR20040086644A - DNA Chip for Diagnosis of Leukemia - Google Patents

Abstract

PURPOSE: A DNA chip for diagnosis of leukemia is provided, which rapidly and accurately discriminates genotype of leukemia, so that it can be useful for diagnosis of leukemia at an early stage. CONSTITUTION: The DNA chip for diagnosis of leukemia comprises (i) gene probes for detecting leukemia specific gene disorder, comprising 18 to 25 nucleotides; (ii) a linker comprising 15 thymines(dTTP), 6 CH2 chains and amine, and linked to the 5'-terminal of the gene probe; and (iii) a solid with aldehyde on its surface and linked to the linker by Schiff's base reaction between the surface aldehyde with the amine of the linker, wherein the gene probes comprise selection probes of SEQ ID NO:1 to SEQ ID NO:23 and comparison probes of SEQ ID NO:24 to SEQ ID NO:31.

Description

DNA Chip for Diagnosing Leukemia {DNA Chip for Diagnosis of Leukemia}

The present invention relates to a DNA chip for diagnosing leukemia. More specifically, the present invention relates to a leukemia diagnostic DNA chip for analyzing leukemia specific gene abnormalities, a method for producing an electrical DNA chip, and a genotyping kit for diagnosing leukemia, including an electrical DNA chip.

Leukemia is a malignant blood disease that occurs in the human hematopoietic system, including hematopoietic stem cells that produce blood cells and lymphocytes. The immature malignant leukocytes, which are caused by disorders in the hematopoietic process in which leukocytes mature, proliferate in the bone marrow. It is a kind of blood cancer that malignant leukocytes occupy more than 30% of all bone marrow hematopoietic stem cells. Leukemia is classified into 4 types according to acute and chronic, myeloid and lymphoid composition, and it is classified into 20 types of leukemia in terms of histological aspects. All types of leukemias can occur at any age, but predominantly in children (2-10 years) is acute lymphocytic leukemia, and what is commonly seen in middle or old age is known as acute myeloid leukemia or chronic myelogenous leukemia. The incidence of leukemia in Korea was 0.96 per 100,000 population in 1992, and between 3,000 and 4,000 people were diagnosed with new leukemia every year, and in 2000, 1,306 people, or 0.54% of all deaths, died from leukemia. Ministry of Health, Cancer, and Cancer Patients Survey Report, 1996; Statistical Yearbook of Causes of Death, 2000.

The known methods of diagnosing leukemia are known to diagnose peripheral blood cells and bone marrow cells by conventional optical microscopy and staining.However, even tumors showing the same findings show very different clinical results depending on the patient and are treated in various treatments. Because of the different responses, the diagnosis and classification according to the pathology, morphological specificity, presents limitations in determining the appropriate treatment for the treatment of leukemia. Recently, molecular genetics has been developed, as a leukemia-specific chromosomal abnormalities and gene identified in more than 80-90% of malignant blood diseases, molecular diagnostics by a chromosomal abnormality is high yeombeop minutes, fluorescence in-situ hybrid method (in situ hybridization) and polymerase chain reaction methods have been used to improve the success rate of diagnosis. Nevertheless, the diagnostic accuracy for determining the type of leukemia is only about 30 to 40%, and the disadvantage that the time required for diagnosis is high is not improved. Therefore, the development of new molecular genetic diagnostics that can more accurately and quickly diagnose various leukemia-specific genes and to determine the prognosis after the treatment of diseases by measuring the presence of microscopic residual disease is urgently needed, but the achievements have yet to be made. It is not reported.

Therefore, there is a constant need to develop a method for analyzing gene abnormalities in leukemia patients quickly and accurately.

Accordingly, the present inventors have made diligent research to develop a method for rapidly and accurately analyzing gene abnormalities of leukemia patients. As a result, the present inventors have collected DNA chips and samples containing probes that can specifically bind to leukemia-specific abnormal genes. Genetic analysis of leukemia patients can be analyzed using leukemia diagnostic genotyping kits, which include primers for reverse transcription of RNA into cDNA and amplification by PCR, and dideoxy nucleotides for fluorescent labeling of amplification products upon amplification. This invention was completed.

After all, the main object of the present invention is to provide a leukemia diagnostic DNA chip for classifying and diagnosing the type of leukemia.

Another object of the present invention is to provide a method for producing an electrical DNA chip.

Still another object of the present invention is to provide a genotyping kit for diagnosing leukemia comprising an electric DNA chip.

1 is a schematic diagram showing the position of the probe in the DNA chip.

Figure 2 is a photograph showing the results of analysis of chronic myelogenous leukemia (CML) caused by the fusion of exon 14 of the BCR gene and exon 2 of the ABL gene.

3 is a photograph showing the results of analyzing CML caused by the fusion of exon 13 of the BCR gene and exon 2 of the ABL gene.

4 is a photograph showing the results of analyzing CML caused by the fusion of exon 1 of the BCR gene and exon 2 of the ABL gene.

5 is a photograph showing the results of analyzing CML caused by the fusion of exon 19 of the BCR gene and exon 2 of the ABL gene.

Figure 6 is a photograph showing the results of analysis of acute myeloid leukemia (AML) caused by the fusion of the AML gene and the ETO1 gene.

Figure 7 is a photograph showing the results of analyzing the AML caused by the fusion of PMLa1 gene and Rara gene.

8 is a photograph showing the results of analyzing AML caused by the fusion of the PMLb1 gene and the Rara gene.

9 is a photograph showing the results of analyzing the AML caused by the fusion of exon 12 of the CBFB gene and MYH gene.

Leukemia diagnostic DNA chip of the present invention is composed of 18 to 25 nucleotides, gene probes for detecting leukemia specific gene abnormalities; A linker comprising 15 thymine (dTTP), 6 CH ₂ chains and an amine group sequentially, wherein the 5 ′ end of the electric gene probe is linked to the thymine site; And a solid linked to an aldehyde on the surface, and an amine group of the electric linker connected through a reaction of the aldehyde on the surface with a Schiff's base: wherein the gene probe is not particularly limited, but is represented by SEQ ID NOs: 1 to 23 It is preferred to use a selection probe and a control probe of SEQ ID NOS: 24-31.

The method for producing a DNA chip for diagnosing electric leukemia is composed of 18 to 25 nucleotides, the method comprising: synthesizing a gene probe for detecting leukemia specific gene abnormalities; Synthesizing a linker comprising 15 thymine (dTTP), 6 CH ₂ chains, and an amine group sequentially; Binding the 5 'end of the electrosynthesized gene probe to the thymine region of the electrosynthesized linker; Binding a gene probe-linked linker to a surface of an aldehyde-bound solid through a Schiff's base reaction; And a step of reducing unreacted aldehyde, wherein the solid is not particularly limited but is preferably glass, and it is preferable to use a reducing agent such as NaBH _{4 although} the reducing conditions of the aldehyde are not particularly limited. Do.

On the other hand, using the leukemia diagnostic DNA chip of the present invention, it is possible to produce a genotyping kit for screening leukemia patients. That is, the leukemia diagnostic genotyping kit of the present invention is a leukemia diagnostic DNA chip, a primer for amplifying RNA from a sample by cDNA and PCR method, and fluorescently labeled dideoxy nucleotides capable of labeling amplification products in a polymerase chain reaction. And lambda exonuclease for isolating the amplification product into single strands: wherein the fluorescently labeled dideoxy nucleotides are not particularly limited, but Texas Red ddATP, Cyanine 3 ) ddCTP, Cyanine 5 ddGTP or fluorescein-12 ddUTP.

The above-described genotyping kit of the present invention can be used to select leukemia patients. That is, synthesizing the cDNA from the RNA collected from the sample to be tested using the primers of the genotyping kit of the present invention, amplifying the synthesized cDNA using a fluorescently labeled dideoxy nucleotides; Separating the amplified cDNA into single strands; Applying a single strand of cDNA to the DNA chip to perform a hybridization reaction; And, the leukemia patients can be selected by washing the DNA chip and using a scanner to examine the probe site on the DNA chip: wherein the primers used to amplify the cDNA are leukemia specific. It is preferable to use a combination of several kinds of primers to amplify all of the above genes, and the fluorescently labeled dideoxy nucleotides are not particularly limited, but it is preferable to use cyanine 5 ddCTP. Although the method of separating into strands is not particularly limited, it is preferable to use a method using an enzyme, and more preferably, lambda exonuclease included in the genotyping kit of the present invention is used.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of DNA Chips

Example 1-1 Construction of a Probe Linked with a Linker

In order to diagnose leukemia specific genes, a probe containing 18 to 22mer oligonucleotides containing translocated gene regions and a region having normal sequences at each gene translocation region was constructed and 15 thymines were used. (dTTP), a linker with six CH ₂ chains and an amine group were constructed sequentially, and then the 5 'end of the electrically constructed probe was connected to the thymine portion of the linker. The base sequence of the probe is as follows:

Screening probe

Probe 1: BA1 5'-GCAGAGTTCAAAAGCCCTTCAG-3 '(SEQ ID NO: 1)

Probe 2: BA2 5'-CCATCAATAAGGAAGAAGCCC-3 '(SEQ ID NO: 2)

Probe 3: BA3 5'-GGAGACGCAGAAGCCCTTC-3 '(SEQ ID NO: 3)

Probe 4: BA4 5'-CCTTCGACGTCAAAGCCCTTCA-3 '(SEQ ID NO: 4)

Probe 5: AEA1 5'-GAGAACCTCGAAAATCATGGATG-3 '(SEQ ID NO: 5)

Probe 6: AMDS 5'-GAACCTCGAAATAATGAGTGTG-3 '(SEQ ID NO: 6)

Probe 7: AET1 5'-GAGAACCTCGAAATCGTACTGA-3 '(SEQ ID NO: 7)

Probe 8: PRa1 5'-GGGGAGGCAGCCATTGAGAC-3 '(SEQ ID NO: 8)

Probe 9: PRb1 5'-CAGGGGAAAGCCATTGAGAC-3 '(SEQ ID NO: 9)

Probe 10: NuRa1 5'-GGGGCCCCTCCAGCCATT-3 '(SEQ ID NO: 10)

Probe 11: PR2 5'-TTACTGGCTCATTCAGCCATTGA-3 '(SEQ ID NO: 11)

Probe 12: CM1 5'-AGGAAATGGAGGAGTTGCTTC-3 '(SEQ ID NO: 12)

Probe 13: CM (e7) 5'-AGGAAATGGAGTTCAAGAGGG-3 '(SEQ ID NO: 13)

Probe 14: CM (e8) 5'-GGAAATGGAGAATGAAGTTGAG-3 '(SEQ ID NO: 14)

Probe 15: CM (e12) 5'-AGGAAATGGAGTCCATGAGCT-3 '(SEQ ID NO: 15)

Probe 16: ME1 5'-CTGTAATCCCGAGAGAATGG-3 '(SEQ ID NO: 16)

Probe 17: ME2 5'-AGTGGACTTTAAGGATTCTGTTTT-3 '(SEQ ID NO: 17)

Probe 18: MA4 5'-CTAAGTGTTAATAGACTTCTGCA-3 '(SEQ ID NO: 18)

Probe 19: A4M2 5'-GTTTGTAAATACTGGCAGGCG-3 '(SEQ ID NO: 19)

Probe 20: MA6 5'-CCAGAATCAGGATTTGGAGTTCCA-3 '(SEQ ID NO: 20)

Probe 21: MA9 5'-GGGCATGTAGAGGACCCTAAT-3 '(SEQ ID NO: 21)

Probe 22: MA10 5'-AGAAACAAAAAAAACATTTTATTTGAA-3 '(SEQ ID NO: 22)

Probe 23: MA17 5'-CAGAATCAGGCATCTACACCAG-3 '(SEQ ID NO: 23)

Control probe

Probe 24: ABL 5'-TATCTGGAAGAAGCCCTTCAGC-3 '(SEQ ID NO: 24)

Probe 25: BCR13 5'-AATAAGGAAGATGATGAGTCTCCG-3 '(SEQ ID NO: 25)

Probe 26: BCR14 5'-AGCAGAGTTCAAATCTGTACTGCAC-3 '(SEQ ID NO: 26)

Probe 27: AML1 5'-GAGAACCTCGAAGACATCGGCA-3 '(SEQ ID NO: 27)

Probe 28: EAP 5'-GTAGAAGATGGAATCATGGATG-3 '(SEQ ID NO: 28)

Probe 29: RARA 5'-CCCCAGCCACCATTGAGAC-3 '(SEQ ID NO: 29)

Probe 30: CBFB 5'-AGGAAATGGAGGTGAGAGTTTC-3 '(SEQ ID NO: 30)

Probe 31: MLL6 5'-CCAGAATCAGGAAAAACCACC-3 '(SEQ ID NO: 31)

Example 1-2 Preparation of DNA Chips

The probes connected to each linker constructed in Example 1-1 were dissolved at 50 μM in buffer (350 mM sodium bicarbonate, pH 9.0), respectively, and slides coated with aldehyde (silylated slide, CSS-100, CEL, Houston, TX, USA) using an arrayer (MicroGrid II, BioRobotics, USA) on the surface was dropped to 150㎛ size, 400㎛ interval, and then the seed base reaction. Subsequently, the mixture was washed sequentially with 0.2% (w / v) SDS solution and tertiary distilled water, and immersed in NaBH ₄ solution (0.1 g NaBH ₄ , 30 ml PBS, 10 ml ethanol) for 15 minutes. The non-aldehyde residue was reduced, washed with tertiary distilled water and dried to prepare a DNA chip (see FIG. 1). 1 is a schematic diagram showing the position of the probe in the DNA chip. Each notation indicated in FIG. 1 means a corresponding probe, and the part marked with + is a marker for identifying the direction of the DNA chip. In this case, a marker was used as a template of a part corresponding to 1082bp to 1932bp of the YPL223c gene located in the chromosome XVI of S. cerevisiae , and amplified by a polymerase chain reaction using cy5-ddCTP. . As shown in FIG. 1, the DNA chip of the present invention was prepared to arrange 23 sorting probes and 8 control probes sequentially on a DNA chip to effectively select abnormal genes that cause each leukemia.

Example 2 Leukemia Specific Gene Selection Using DNA Chip

Example 2-1 Obtaining RNA and cDNA Synthesis

Lymphocytes from blood were separated by density gradient centrifugation using Ficoll-Hypaque, and total RNA was obtained from TRI solution (TRI Reagent, Molecular Research Center, INC.) From the isolated lymphocytes. It was. Totally obtained 8 ug of RNA, 100 units reverse transcriptase (M-MLV Reverse Transcriptase, Ambion, USA), 20 units superase (SUPERase in ^TM , Ambion, USA), 100 μM random primer (random 9mer primer), 2.5 mM dNTP (Roche) , USA), 5 minutes at 65 ° C, 5 minutes at 4 ° C, 2 hours at 38 ° C, 5 minutes at 99 ° C and 10 minutes at 4 ° C using 10X first strand synthesis buffer and DEPC-distilled water. By reacting, cDNA was synthesized.

Example 2-2 Amplification and Processing of cDNA

5 μl of electrosynthesized cDNA, 5 μl of 10X PCR buffer, 2 μm of 2.5 mM ddAGT (2.5 mM ddATP, 2.5 mM ddGTP, 2.5 mM ddTTP), 5 μl of 0.1 mM ddCTP, 5 μm of 0.1 cyanine 5 (Cyanine 5) ddCTP 5 μl After mixing 2.5units of Taq polymerase, primers, and adding 50 μl of tertiary distilled water, cDNA was amplified by polymerase chain reaction. At this time, the base sequence of the primer used is as follows, double sense primer contains a phosphate (phosphate) in the 5 'direction.

CML Related Sense Primer

ABL-F: 5'-GTTGGAGATCTGCCTGAAGC-3 '(SEQ ID NO: 32)

BCR13 / 14-F: 5'-GATGCTGACCAACTCGTGTG-3 '(SEQ ID NO: 33)

AML1-F: 5'-TCGAAGTGGAAGAGGGAAAA-3 '(SEQ ID NO: 34)

EAP-F: 5'-CCATGGCTCCTGTGAAAAAG-3 '(SEQ ID NO: 35)

BCR1-F: 5'-GCAGAACTCGCAACAGTCCT-3 '(SEQ ID NO: 36)

BCR19-F: 5'-TCGGAGTCAAGATTGCTGTG-3 '(SEQ ID NO: 37)

CML related antisense primer

ABL-R: 5'-AACGAAAAGGTTGGGGTCAT-3 '(SEQ ID NO: 38)

BCR13 / 14-R: 5'-CAGCTGTGTCCCTGTAGACG-3 '(SEQ ID NO: 39)

AML1-R: 5'-GCTCGGAAAAGGACAAGCTC-3 '(SEQ ID NO: 40)

EAP-R: 5'-TTGCTCTTGCTCCTTTCGAT-3 '(SEQ ID NO: 41)

Sense primer set 2 related to AML : RARA-F, PMLa1-F, PMLb1-F, PLZF-F, NuMA-F

RARA-F: 5'-CCTCCCTACGCCTTCTTCTT-3 '(SEQ ID NO: 42)

PMLa1-F: 5'-CAAGAAAGCCAGCCCAGAG-3 '(SEQ ID NO: 43)

PMLb1-F: 5'-GTGCGCCAGGTGGTAGCTC-3 '(SEQ ID NO: 44)

PLZF-F: 5'-CCACAAGGCTGACGCTGTATT-3 '(SEQ ID NO 45)

NuMA-F: 5'-CGAGCCACCTCCTCTACTCA-3 '(SEQ ID NO: 46)

Antisense Primer Set 2 related to AML : RARA-R

RARA-R: 5'-GCTTGTAGATGCGGGGTAGA-3 '(SEQ ID NO: 47)

Sense primer set 3 related to AML : AML1-F, CBFB-F, MLL6-F

AML1-F: 5'-TCGAAGTGGAAGAGGGAAAA-3 '(SEQ ID NO: 34)

CBFB-F: 5'-TTTGAAGAGGCTCGGAGAAG-3 '(SEQ ID NO 48)

MLL6-F: 5'-GAGCCCAAGAAAAAGCAGCC-3 '(SEQ ID NO 49)

Antisense primer set 3 related to AML : CBFB-R, MLL6-R, ETO-R, MDS-R

CBFB-R: 5'-GTAAAGATGGGCAGCACACA-3 '(SEQ ID NO: 50)

MLL6-R: 5'-ATCCTGTGGACTCCATCTGC-3 '(SEQ ID NO: 51)

ETO-R: 5'-TTGAGTAGTTGGGGGAGGTG-3 '(SEQ ID NO: 52)

MDS-R: 5'-CGATCTTCCTTTTGGTCCATATTC-3 '(SEQ ID NO: 53)

Sense primer set 4 related to AML : CBFB-F

CBFB-F: 5'-TTTGAAGAGGCTCGGAGAAG-3 '(SEQ ID NO: 54)

Antisense primer set 4 related to AML : CBFB-R, MYH11-R, MYH11 (e12) -R, MYH11 (e7 / e8) -R

CBFB-R: 5'-GTAAAGATGGGCAGCACACA-3 '(SEQ ID NO: 50)

MYH11-R: 5'-GCAGCTTCGTAGACACGTTG-3 '(SEQ ID NO: 55)

MYH11 (e12) -R: 5'-ATCCCTGTGACGCTCTCAAC-3 '(SEQ ID NO: 56)

MYH11 (e7 / e8) -R: 5'-CATCTCCTCCATCTGGGTCT-3 '(SEQ ID NO: 57)

Subsequently, the amplified cDNA was treated with lambda exonuclease (10 units / μl) to make single-stranded DNA.

Example 2-3 : Hybridization and Confirmation Using DNA Chips

CDNA separated into single strands was applied to the DNA chip prepared in Example 1-2, and the hybridization reaction was performed at 45 ° C. for 1 hour. Subsequently, the DNA chip in which the hybridization reaction is completed is washed twice at room temperature with washing solution I (0.1% SDS, 2X SSC) for 5 minutes, and once at room temperature with washing solution II (0.2X SSC), and then washing solution III Washed once with (0.1X SSC) at room temperature for 5 minutes, dried at room temperature, and then scanned with a DNA chip dried at a wavelength of 667 nm in the cyanine 5 ddCTP wavelength range (see FIGS. 2 to 9). ). 2 to 9 are abnormalities of BCR genes found in chronic myelogenous leukemia (CML) and AML genes found in acute myeloid leukemia (AML), ETO1 gene, PMLa1 gene, using the leukemia diagnostic genotyping kit of the present invention. Abnormalities of the Rara gene, CBFB gene and MYH gene are detected.

Figure 2 is a photograph showing the results of analyzing the CML caused by the fusion of exon 14 of the BCR gene and exon 2 of the ABL gene, cDNA amplified using the sense primer set 1 and antisense primer set 1 associated with the CML It was found that the CML-related control probes 24, 25, 26, 27 and 28 and the screening probe 1 were positive.

Figure 3 is a photograph showing the results of analyzing the CML caused by the fusion of exon 13 of the BCR gene and exon 2 of the ABL gene, cDNA amplified using the sense primer set 1 and antisense primer set 1 associated with the CML It was found that the CML-related control probes 24, 25, 26, 27 and 28 and the screening probe 2 were positive.

Figure 4 is a photograph showing the results of analyzing the CML caused by the fusion of exon 1 of the BCR gene and exon 2 of the ABL gene, cDNA amplified using the sense primer set 1 and antisense primer set 1 associated with the CML It was found that the control probes related to CML were positive in 24, 25, 26, 27 and 28 and the selection probe 3.

5 is a photograph showing the results of analyzing the CML caused by the fusion of exon 19 of the BCR gene and exon 2 of the ABL gene, and cDNA amplified using the sense primer set 1 and the antisense primer set 1 associated with the CML. The control probes related to CML, 24, 25, 26, 27 and 28 and the screening probe 4 was positive.

Figure 6 is a photograph showing the results of analyzing the AML caused by the fusion of the AML gene and the ETO1 gene, amplified using the sense primer set 2 and antisense primer set 2, sense primer set 3 and antisense primer set 3 associated with AML cDNA was positive in AML-related control probes 29, 30 and 31 and screening probe 7.

7 is a photograph showing the results of analyzing the AML caused by the fusion of PMLa1 gene and Rara gene, amplified using the sense primer set 2 and antisense primer set 2, sense primer set 3 and antisense primer set 3 associated with AML cDNA was positive in AML-related control probes 29, 30 and 31 and screening probe 8.

FIG. 8 is a photograph showing analysis results of AML caused by fusion of PMLb1 gene and Rara gene, and amplified using sense primer set 2, antisense primer set 2, sense primer set 3, and antisense primer set 3 related to AML. cDNA was positive in AML-related control probes 29, 30 and 31 and screening probe 9.

9 is a photograph showing the results of analysis of AML caused by the fusion of exon 12 of the CBFB gene and MYH gene, wherein cDNA amplified using sense primer set 4 and antisense primer set 4 related to AML is related to AML. It was found that positive reaction was observed in the number 30 probe and the number 15 screening probe.

As can be seen from the above results, using the leukemia diagnostic genotyping kit of the present invention, it can be seen that the gene abnormalities of each leukemia can be specifically selected.

As described and demonstrated in detail above, the present invention provides a leukemia diagnostic DNA chip for analyzing leukemia specific gene abnormalities, a method for producing an electrical DNA chip, and a genotyping kit for diagnosing leukemia, including an electrical DNA chip. Leukemia diagnostic DNA chip of the present invention is composed of 18 to 25 nucleotides, gene probes for detecting leukemia specific gene abnormalities; A linker comprising 15 thymine (dTTP), 6 CH ₂ chains and an amine group sequentially, wherein the 5 ′ end of the electric gene probe is linked to the thymine site; And, the aldehyde is bonded to the surface, and the amine group of the electric linker includes a solid connected through the aldehyde group and the Schiff base (Schiff's base) reaction of the surface. By using the genotyping kit of the present invention, the genotype of leukemia can be distinguished quickly and accurately, and thus will be widely used for early diagnosis of leukemia.

As described above in detail the specific parts of the present invention, for those skilled in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> DISGENE          KIM, Dong Wook <120> Genotyping Kit for Diagnosis of Leukemia <160> 57 <170> KopatentIn 1.6 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe BA1 <400> 1 gcagagttca aaagcccttc ag 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe BA2 <400> 2 ccatcaataa ggaagaagcc c 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe BA3 <400> 3 ggagacgcag aagcccttc 19 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe BA4 <400> 4 ccttcgacgt caaagccctt ca 22 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe AEA1 <400> 5 gagaacctcg aaaatcatgg atg 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe AMDS <400> 6 gaacctcgaa ataatgagtg tg 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe AET1 <400> 7 gagaacctcg aaatcgtact ga 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe PRa1 <400> 8 ggggaggcag ccattgagac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe PRb1 <400> 9 caggggaaag ccattgagac 20 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe NuRa1 <400> 10 ggggcccctc cagccatt 18 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe PR2 <400> 11 ttactggctc attcagccat tga 23 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe CM1 <400> 12 aggaaatgga ggagttgctt c 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe CM (e7) <400> 13 aggaaatgga gttcaagagg g 21 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe CM (e8) <400> 14 ggaaatggag aatgaagttg ag 22 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe CM (e12) <400> 15 aggaaatgga gtccatgagc t 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe ME1 <400> 16 ctgtaatccc gagagaatgg 20 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe ME2 <400> 17 agtggacttt aaggattctg tttt 24 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe MA4 <400> 18 ctaagtgtta atagacttct gca 23 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe A4M2 <400> 19 gtttgtaaat actggcaggc g 21 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe MA6 <400> 20 ccagaatcag gatttggagt tcca 24 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe MA9 <400> 21 gggcatgtag aggaccctaa t 21 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> probe MA10 <400> 22 agaaacaaaa aaaacatttt atttgaa 27 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe MA17 <400> 23 cagaatcagg catctacacc ag 22 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe ABL <400> 24 tatctggaag aagcccttca gc 22 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe BCR13 <400> 25 aataaggaag atgatgagtc tccg 24 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe BCR14 <400> 26 agcagagttc aaatctgtac tgcac 25 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe AML1 <400> 27 gagaacctcg aagacatcgg ca 22 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe EAP <400> 28 gtagaagatg gaatcatgga tg 22 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe RARA <400> 29 ccccagccac cattgagac 19 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe CBFB <400> 30 aggaaatgga ggtgagagtt tc 22 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe MLL6 <400> 31 ccagaatcag gaaaaaccac c 21 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe ABL-F <400> 32 gttggagatc tgcctgaagc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe BCR13 / 14-F <400> 33 gatgctgacc aactcgtgtg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe AML1-F <400> 34 tcgaagtgga agagggaaaa 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe EAP-F <400> 35 ccatggctcc tgtgaaaaag 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe BCR1-F <400> 36 gcagaactcg caacagtcct 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe BCR19-F <400> 37 tcggagtcaa gattgctgtg 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe ABL-R <400> 38 aacgaaaagg ttggggtcat 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe BCR13 / 14-R <400> 39 cagctgtgtc cctgtagacg 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe AML1-R <400> 40 gctcggaaaa ggacaagctc 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe EAP-R <400> 41 ttgctcttgc tcctttcgat 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe RARA-F <400> 42 cctccctacg ccttcttctt 20 <210> 43 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe PMLa1-F <400> 43 caagaaagcc agcccagag 19 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe PMLb1-F <400> 44 gtgcgccagg tggtagctc 19 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe PLZF-F <400> 45 ccacaaggct gacgctgtat t 21 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe NuMA-F <400> 46 cgagccacct cctctactca 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe RARA-R <400> 47 gcttgtagat gcggggtaga 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe CBFB-F <400> 48 tttgaagagg ctcggagaag 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe MLL6-F <400> 49 gagcccaaga aaaagcagcc 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe CBFB-R <400> 50 gtaaagatgg gcagcacaca 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe MLL6-R <400> 51 atcctgtgga ctccatctgc 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe ETO-R <400> 52 ttgagtagtt gggggaggtg 20 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe MDS-R <400> 53 cgatcttcct tttggtccat attc 24 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe CBFB-F <400> 54 tttgaagagg ctcggagaag 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe MYH11-R <400> 55 gcagcttcgt agacacgttg 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe MYH11 (e12) -R <400> 56 atccctgtga cgctctcaac 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe MYH11 (e7 / e8) -R <400> 57 catctcctcc atctgggtct 20

Claims (6)

(Iii) a gene probe consisting of 18 to 25 nucleotides, for detecting leukemia specific gene abnormalities; (Ii) a linker comprising 15 thymine (dTTP), 6 CH ₂ chains and an amine group sequentially, wherein the 5 ′ end of the electric gene probe is linked to the thymine moiety; And, (Iii) An aldehyde coupled to the surface, the amine group of the electric linker comprising a solid linked to the aldehyde group and the Schiff base (Schiff's base) reaction of the surface, leukemia diagnostic DNA chip. The method of claim 1, Gene probes are characterized in that the selection probe of SEQ ID NO: 1 to 23 and the control probe of SEQ ID NO: 24 to 31 DNA chip for leukemia diagnosis. (Iii) synthesizing a gene probe consisting of 18 to 25 nucleotides to detect leukemia specific gene abnormalities; (Ii) synthesizing a linker sequentially comprising 15 thymine (dTTP), 6 CH ₂ chains and an amine group; (Iii) binding the 5 'end of the electrosynthesized gene probe to the thymine site of the electrosynthesized linker; (Iii) binding the linker to which the gene probe is bound to the surface of the aldehyde-bound solid through a Schiff's base reaction; And, (Iii) A method for producing a leukemia diagnostic DNA chip, comprising the step of reducing aldehyde remaining without reacting. The DNA chip of claim 1, a primer for amplifying RNA obtained from a sample by cDNA and PCR method, a fluorescently labeled dideoxy nucleotide capable of labeling amplification products in a polymerase chain reaction, and a single strand for separating amplification products. Genotyping kit for diagnosing leukemia, including lambda exonuclease. The method of claim 4, wherein Primer is characterized in that the antisense primer or sense primer having a nucleotide sequence of SEQ ID NO: 32 to 57 Genotyping kit for diagnosing leukemia. The method of claim 4, wherein Fluorescently labeled dideoxy nucleotides are characterized as Texas Red ddATP, cyanine 3 ddCTP, cyanine 5 ddGTP or fluorescein-12 ddUTP. Genotyping kit for diagnosing leukemia.