KR101019952B1 - Stroke early diagnosing chip and screening analysis thereof - Google Patents

Abstract

Stroke is a disease confronting the cause of death due to a single disease at home and abroad, and active treatment is required, but the current diagnosis method remains within the scope of etiological diagnosis for selecting a treatment direction for acute stroke.

Accordingly, the present invention provides a high-throughput analysis method capable of early diagnosis of stroke expression potential through non-invasive diagnosis, stroke expression prediction gene marker SNP analysis and biochemical marker analysis through a small amount of blood collection.

Conventional SNP analysis based on single base extension (SBE) has been reported to reduce discrimination in some base conversions, and has the disadvantage of relatively low throughput and long analysis time compared to the chip method. do. Accordingly, the present invention is designed to position the SNP base complementary to the target gene in the central (central) of the probe, and to design the probe so that 5 to 6 relatively short bases to the left and right, and the detection discrimination ability for base substitution, insertion, deletion Provides an assay with improved specificity.

Stroke, DNA chip, PNA chip, early diagnosis, single nucleotide polymorphism (SNP), gene markers, probes, alleles.

Description

Stroke early diagnosing chip and screening analysis

The present invention relates to a chip for early diagnosis of stroke, and more specifically, using allele-specific probes for the presence or absence of single nucleotide polymorphism (SNP) of genes for predictive analysis of acute ischemic and hemorrhagic stroke disease expression. And a method for discriminating in a high-throughput manner.

Stroke is largely divided into ischemic stroke (cerebral infarction) due to central nervous system vascular occlusion that supplies blood to the brain and hemorrhagic stroke due to vascular rupture. Cerebral infarction (ischemic stroke) is the most common among stroke patients, followed by cerebral hemorrhage and subarachnoid hemorrhage. Ischemic strokes in which blood vessels are blocked by arteriosclerosis or thrombus require thrombolytics to penetrate the blocked blood vessels, which can reduce mortality and sequelae intensity. Hematoma should be removed while preventing the necrosis of brain tissue. The clinical findings of stroke are accompanied by severe sequelae such as paraplegia, sensory, speech and vision disorders and dementia. In recent years, the treatment technology for acute cerebral ischemia has developed a lot. Recombinant tissue plasminogen activator (rt-PA), a jugular vein thrombolytic agent, has been approved by the US FDA as a treatment for acute cerebral ischemia within 3 hours of onset. In addition, carotid endarterectomy (CEA) has been proven to prevent recurrence of cerebral infarction in patients with extracranial carotid artery.

Reported by the World Health Organization in 2002, the number of deaths from cardiovascular disease worldwide was 16.7 million, with coronary artery disease and stroke accounting for 7.2 million and 5.5 million deaths, respectively. 76% of all deaths were related to disease (http://www.who.int/cardiovascular_diseases/)

resources / atlas / en /). In the United States, 4.5 million stroke patients are reported, 600,000 patients die each year, 770,000 deaths are reported, and medical expenses of $ 50 billion are required annually. In Korea, according to the 2000 statistics, stroke is competing for the death level following cancer, and the number of patients is estimated to be about 1 million.

As described above, CT and computer aided tomography (MRI) or magnetic resonance imaging (MRI) for the pathological diagnosis of dichotomous hemorrhagic or infarct due to extremely contradictory treatment mechanisms of ischemic stroke and hemorrhagic stroke. magnetic resonance imaging), and in addition, the measurement of cerebrovascular flow using radioisotopes or ultrasound, cerebrovascular imaging, and the like are possible. CT is most helpful for the differential diagnosis between ischemic stroke and hemorrhagic stroke, and MRI has been evaluated to be excellent for determining ischemic stroke and vascular status in acute stage. However, there are disadvantages such as high cost and limitation of diagnosis site due to the use of expensive equipment. In addition, local diagnosis to determine exactly where the lesion is located, including the cerebral cortex, is also important for treatment and prognostic evaluation.

Brain tissues do not regenerate once damaged and the sequelae is severe once they occur, so the importance of early diagnosis for stroke prevention, as opposed to the diagnosis for treatment decisions after such an outbreak, is socioeconomic In addition to the reduction, the net functional value is very high in terms of mortality reduction and national disease management. It is urgently needed to prevent proactive prevention through the development of early diagnosis technology for stroke and the establishment of a risk factor medical management system that analyzes the possibility of stroke and controls risk factors accordingly.

Therefore, the present invention is to provide an analysis method for early diagnosis of the possibility of stroke expression. In detail, the present invention is to provide a test method that can realize the purpose of the early diagnosis of stroke according to the comprehensive analysis results of four biochemical markers for phenotypic disease predisposition analysis and 11 genetic markers for genetic predisposition analysis. In order to realize the object of the present invention, to provide a DNA or PNA chip that can easily analyze the early diagnosis of stroke through the allele-specific hybridization reaction results of 11 gene markers in one response. .

Although stroke is the number one mortality disease among the single diseases, the current diagnostic method is a differential diagnosis of whether the stroke etiology is ischemic or hemorrhagic and has the characteristics of diagnosis to select a treatment direction after the onset of the disease. The present invention aims to contribute to the reduction of incidence, mortality, and alleviation of sequelae disorders through early diagnosis of stroke by analyzing the presence or absence of SNPs of four biochemical markers and 11 stroke expression predictive genes in a small amount of blood.

The pathophysiological events related to the cardiovascular and cerebrovascular vessels were analyzed by measuring four biochemical markers, and the SNP analysis of the 11 genes for predictive analysis of the genetic disposition related to stroke expression was analyzed. We will provide analytical methods and chips that enable early diagnosis of stroke by evaluating and integrating them.

Conventional SNP analysis, based on single base extension (SBE), reduces discrimination for some base conversions (eg, A> G substitution), and provides relatively low throughput, There are disadvantages of long analysis time. Therefore, the present invention is to design an allele-specific probe to position the SNP base complementary to the target gene in the central (central) of the probe, and 5-6 relatively short bases to the left and right to determine the SNP discrimination and specificity I want to improve.

In order to achieve the object of the present invention, there is provided an analytical method capable of early diagnosis of stroke through single-nucleotide polymorphism (SNP) high-throughput analysis and biochemical marker analysis of stroke expression predictive gene markers.

In detail, the analysis method comprising the steps of: fabricating a chip (spot) spotted with a probe modified with an amino modifier or thiol modifier at the 5'- or 3'-end;

Isolating genomic DNA from blood;

Preparing a target DNA amplification product to include fluorescent dyes while amplifying probe target sites of stroke expression predictive marker genes through multiplex polymerase chain reaction (PCR);

Performing a hybridization reaction between the multiplex PCR reaction product and a probe immobilized on a chip surface and washing the hybridization reaction;

Analyzing SNP results of stroke expression predictive markers by scanning a chip with a laser of a wavelength specific to fluorescent dyes and measuring fluorescence intensity according to the hybridization reaction result;

Analyzing and integrating SNP results and biochemical marker test results of the stroke expression predictive marker genes to derive a disease expression predisposition; And

It provides a method for the early diagnosis of stroke comprising the entire analysis step.

In addition, in the above analysis method, a single nucleotide polymorphism (SNP) associated with stroke expression predictive gene markers and their stroke generation mechanism,

MTHFR (5,10-methylenetetrahydrofolate reductase) C677T;

Angiotensinogen Met235Thr;

Apolipoprotein E (APOE) ε2, ε3, ε4;

Coagulation factor V Leiden (F5) Arg506Gln;

Coagulation factor II prothrombin (F2) G20210A;

Fibrinogen G (-455) A;

Glycoprotein Ib (platelet alpha polypeptide) T (-5) C;

Fatty acid-binding protein 2 (FABP2) Ala54Thr;

Plasminogen activator inhibitor-1 (PAI-1) 5G / 4G;

IL-6 (Interleukin-6) G (-174) C;

Nitric oxide synthase 3 (NOS3) Glu298Asp; One or more selected from; And

An internal control provides a method for the early diagnosis of stroke comprising a human beta-globin gene.

In the above analysis method, at least one DNA or PNA probe selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 1 to 49 in order to realize the object of the present invention preferably Provides a chip for early diagnosis of stroke.

The present invention enables early diagnosis of stroke expression potential through a non-invasive sample collection through a small amount of blood collection, a total of 11 types of stroke expression prediction gene marker SNP analysis and 4 types of biochemical marker analysis through high-throughput analysis.

Conventional diagnosis related to stroke has a differential diagnosis of the etiology for selecting a treatment direction after disease expression, but the present invention provides an analysis method and chip capable of prophylactic early diagnosis through biochemical markers and genetic predisposition analysis. Diagnosis is possible regardless of the place and time of diagnosis due to the use of large expensive equipment, and low-cost diagnosis is possible.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings and tables. Configurations shown in the drawings, tables and examples described herein are only one of the most preferred embodiments of the present invention and do not represent all of the technical idea of the present invention, various modifications that can be substituted for them at the time of the present application It should be understood that there may be equivalents and variations.

Also, within the scope of the present invention, various modifications may be made by those skilled in the art to which the present invention pertains, and such modifications are within the scope of the present invention.

< Example  1> Analysis of biochemical markers for clinical diagnosis of stroke

Biochemical markers for the clinical diagnosis of acute cerebral ischemia according to an embodiment of the present invention are matrix metalloproteinase-9 (MMP-9), metalloproteinase tissue inhibitor-1 (tissue inhibitor of metalloproteinase-1, TIMP-1), high-sensitivity C-reactive protein (hs-CRP), and homocysteine. MMP-9 is an endopeptidase that breaks down type IV collagen, laminin and fibronectin, which are the major components of the basal lamina around the cerebrovascular vessels. TIMP-1 is involved in the damage and repair of the brain and peripheral nerves by acting as a counter-regulator that inhibits them by forming complexes with MMPs. MMPs and TIMPs are acute ischemic. There is a study that linked the pathophysiology of stroke (Horstmann S et. al . Stroke (2003) 34: 2165-70, Orbe J et al . Atherosclerosis (2003) 170: 269-276). hs-CRP is an inflammatory marker and is known as an independent predictor of atherosclerosis and cardiovascular pathophysiology at all levels. High concentrations of homocysteine in blood are also known to be associated with stroke, Alzheimer's disease.

Peripheral blood samples were collected from 30 patients diagnosed with MRI, 17 patients with hemorrhagic stroke, and 12 healthy adults without a history of brain disease. Human MMP-9 immunofluorescence (Cat. No. DMP900, R & D systems Inc. Minneapolis, MN, USA) test was performed according to the manufacturer's protocol as follows. 100 uL of assay dilution RD1-34 was dispensed into the corresponding wells of the microplate. 100 uL of each of the standard, control and diluted specimens was added to the wells and allowed to react at room temperature for 2 hours at 500 rpm rotation in a horizontal rotating microplate shaker. The reaction solution was collected and removed, washed with the addition of 400 uL of wash buffer, and then the buffer was removed. The washing process was repeated four times. In the final washing step, after removing the buffer solution, the microplate was inverted onto a paper towel to completely remove the solution. 200 uL of MMP-9 conjugate was added to the wells, and each reaction well was covered with an adhesive strip, followed by 1 hour room temperature reaction on a shaker under the same conditions as above. Since then, a total of four washes were performed under the same conditions. 200 uL of substrate solution was added to each well and allowed to react for 30 minutes at room temperature under light shielding conditions. After the reaction, 50 uL of the reaction stop solution was added to each well, and within 30 minutes, the MMP-9 concentration was measured by measuring the absorbance at 450 nm through a Luminex or Biorad analyzer, and the value was corrected by a 540 or 570 nm wavelength measurement. Human TIMP-1 biochemical marker testing was performed using a human TIMP multiplex kit (Cat. No. LKT003, R & D systems Inc. Minneapolis, MN, USA) according to the manufacturer's protocol. Hs-CRP was measured using an automated chemistry analyzer Hitachi 7600 (Hitachi, Tokyo, Japan), and homocysteine was measured using an automated immunoassay device IMx (Abott Diagnostics, Abbott Park, IL, USA). Total homocysteine was determined by competitive fluorescence polarized immunoassay (FPIA), including unbound, bound, oxidized and reduced.

Comparison of Four Groups of Stroke Biochemical Markers

Ischemic
Stroke group
(n = 30)
P value Hemorrhagic
Stroke group
(n = 17)
P value Normal control
group
(n = 12)
P value MMP-9
(ng / mL)

115.2 (± 132.2)
0.05
112.5 (± 125.4)
0.07
105.3 (± 102.3)
0.06 TIMP-1
(ng / mL)

132.5 (± 83.4)

0.35

127.2 (± 82.2)

0.15

128.4 (± 83.4)

0.08
hs-CRP
(mg / dL)

0.39 (± 0.68)

0.15

0.35 (± 0.64)

0.22

0.32 (± 0.42)

0.17
homocysteine
(μmol / L)

9.8 (± 5.2)

0.37

9.3 (± 5.6)

0.46

8.7 (± 3.7)

0.4

< Example  2> From clinical specimen Genomic DNA  detach

Genomic DNA was extracted from the peripheral blood sample by magnetic bead method. Peripheral blood of 0.03-0.05 mL was added to 96-well or 384-well plates by number of samples and placed in automated nucleic acid extraction equipment. An 8-channel pipetting head is then fitted with a pipette of a pipette tip rack and then aspirates 500 uL of a buffer mix to dissolve the sample, so that each well of the lysis plate It was added to and reacted for 10 minutes. The reaction was promoted by mixing the reaction solution up and down the pipette 15 times during the reaction. Thereafter, 70 uL of the reaction solution containing the magnetic beads was added, mixed with a pipette, and allowed to stand at room temperature for 1 minute, and then a magnetic separator was operated and reacted for an additional 2 minutes. The supernatant except the beads was eluted with a waste reservoir and the magnetic separator was shut down. Thereafter, 500 uL of lysis buffer containing no protease K was added and mixed with a pipette to uniformly spread the magnetic beads. After adding 50 uL of the purification buffer and mixing with a pipette, the mixture was allowed to stand at room temperature for 1 minute, and then a magnetic separator was operated and reacted for another 2 minutes. As above, the supernatant except the beads was eluted with a waste reservoir and the magnetic separator was stopped. Thereafter, 500 uL of washing buffer was added and the magnetic separator was operated twice to remove impurities. In order to recover the DNA as a final procedure, 100 uL of the elution buffer was loaded and pipetted up and down for 5 minutes at room temperature with 50 repetitions, and the supernatant containing pure DNA was recovered and stored until multiplex PCR reaction. .

< Example  3> multiplex PCR Early diagnosis chip through stroke ( chip )of target  Gene amplification

Prior to high-throughput chip analysis, 11 target genes were amplified by multiplex PCR reactions with genomic DNA isolated from the sample as a template. Thereafter, hybridization reactions are performed with selective probes immobilized on the chip, thereby providing genetic predisposition data for early detection of stroke. Multiplex PCR was performed in which 11 genes including angiotensinogen were specifically amplified in a single reaction in the same PCR tube. Multiplexed amplicons were designed to have a short length within 60-200 base pairs to determine the SNP genotype of the genes by forming a duplex with DNA or PNA probes on the chip surface. Electric multiplex PCR includes a human beta-globin gene as an internal control. The compositional composition and PCR reaction conditions of the pre-made PCR mastermix of the 11 gene multiplex PCR described in this Example are shown in Table 2 below.

11 Multiplex PCR Mastermix Composition and PCR Conditions for Early Diagnosis of Stroke
PCR reaction solution composition

Volume (uL)
PCR reaction conditions

Deionized Tertiary Sterilized Distilled Water

4.2

Initial denaturation
(Initial denaturation)
95 ° C., 15 minutes
1 time
11 kinds of primers
Prime premix


5.8


denaturalization
(Denaturation)

95 ° C, 40 seconds




Episode 37




2X Multiplex PCR Premix

15

Combination
(Annealing)
57 ° C, 40 seconds
Template DNA (more than 50 ng)

5

extension
(Extension)
72 ° C., 40 seconds
Final volume

30

Last extension
(Extension)
72 ° C., 7 minutes
1 time

Final concentration of PCR buffer in the multiplex PCR reaction mixture is 50 mM KCl, 3.5 mM MgCl ₂ , 10 mM Tris-HCl, pH 8.2, 2.5 unit Taq polymerase, 300 uM dNTPs (Boehringer Mannheim, Mannheim, Germany), 10 mg / mL Bovine serum albumin. Sense primers of the individual genes included in the 11 primer premixes for early diagnosis of stroke are synthesized by labeling Cy3 or Cy5 fluorescent dyes at the 5'- or 3'-end, or without modification at both ends. Synthesized with nucleotides (Integrated DNA Technologies, Inc., Coralville, IA, USA).

In a multiplex PCR kit including a primer having no fluorescent dye modification at the terminal according to a preferred embodiment of the present invention, Cy3 fluorescent dye-labeled deoxycytosine triphosphate (Cy3-dCTP) constitutes an amplification product during PCR. By adding to the nucleotides to improve the sensitivity and specificity of the reaction. The PCR buffer used for 11 multiplex PCR reactions using Cy3-dCTP had a final concentration of 50 mM KCl, 3.5 mM MgCl ₂ , 10 mM Tris-HCl, pH 8.2, 2.5 units of Taq polymerase, 300 uM dATP , 300 uM dGTP, 300 uM dTTP, 25 uM dCTP, 275 uM Cy3-dCTP (FluoroLink Cy3-dCTP, Amersham Pharmacia Biotech AB, Piscataway, NJ, USA), 10 mg / mL Bovine serum albumin Target genes were specifically amplified.

< Example  4> Early diagnosis chip for stroke chip Make up) Probe ( probe A) design and synthesis

In order to provide a chip for early diagnosis of stroke for realizing the present invention, probes capable of discriminating allele-specific single nucleotide polymorphism (SNP) of 11 genes were designed. .

Specifically, the probes included in the pre-stroke DNA or PNA chip according to a preferred embodiment of the present invention, alleles of a single base polymorphism (SNP) of a total of 11 genes including angiotensinogen In order to discriminate specifically, the fluorescent signal from the duplexes between the PCR amplification product and the probe that exactly matches the SNP base of the allele is located by allowing the SNP corresponding base to be located centrally in the probe sequence. SNP discrimining power was secured by detecting. When the SNP base of the probe is mismatched with the SNP base of the allele complementary to that present in the PCR amplicon, the duplex secondary structure becomes unstable and is removed in the washing process after the chip reaction, thereby having high discrimination ability. Additional factors that can be considered to improve the discrimination ability of the probe include the length of the probe, the stringency of the chip cleaning process, and the chip hybridization reaction temperature. The most important factor to prevent false positive and false negative results was the length of the probe and good discrimination in the 9-15 base pair length range (see FIG. 6).

Primer premier version 5 to select specific probe target sites in the amplicon of multiplex PCR performed for SNP analysis of a total of 11 genes including angiotensinogen , Premier Biosoft International, Palo Alto, CA, USA, DNASIS MAX Version 2.7, MiraiBio Group, South San Francisco, CA, USA, or OligoArray 2.0, http: //cbr-rbc.nrc -cnrc.gc.ca) is designed by applying the program and the details are shown in Table 3. Probes modified to place amine or thiol groups at the 5 'or 3' end of the DNA or PNA probe sequence of interest were synthesized (MWG-Biotech AG, Ebersberg, Germany). When modifying the amino modifier at the 5 'end of the probe, spacers of 6 to 9 carbons or 12 to 15 thymine bases were added to promote the reaction efficiency. No spacer was used when modifying the amino modifier at the 3 'end of the probe. The amine groups of the probe have the properties of primary amine groups and are attached to the aldehyde-activated or carboxyl-activated chip surface. The concentration of the probe was converted to absorbance values at 260 nm, and it was checked for impurities by Maldi-TOF (MALDI-TOF, Matrix Assisted Laser Desorption / Ionization Time-of-Flight), and high performance liquid chromatography (HPLC) After pure purification through liquid chromatography) was prepared so that the final concentration in sterile tertiary distilled water 100-250 pM.

Probe combinations that make up the chip for early diagnosis of stroke order
List
number Target gene symbol name,
Target gene location,
SNP allele name Allele target SNP base of the probe,
Probe direction,
Probe sequence (5'-3 ') Probe
Length
(mer) One MTHFR, exon4, C677T 677C, forward, CGGGAGCCGATTT 13 2 MTHFR, exon4, C677T 677T, forward, CGGGAGTCGATTT 13 3 MTHFR, exon4, C677T 677A, forward, CGGGAGACGATTT 13 4 MTHFR, exon4, C677T 677G, forward, CGGGAGGCGATTT 13 5 AGT, exon2, Met235Thr 704T, forward, TGTCCATGGTG 11 6 AGT, exon2, Met235Thr 704C, forward, TGTCCACGGTG 11 7 AGT, exon2, Met235Thr 704A, forward, TGTCCAAGGTG 11 8 AGT, exon2, Met235Thr 704G, forward, TGTCCAGGGTG 11 9 APOE, exon3, Cys112Arg 336T, reverse, GCCGCACACGT 11 10 APOE, exon3, Cys112Arg 336C, reverse, GCCGCGCACGT 11 11 APOE, exon3, Cys112Arg 336G, reverse, GCCGCCCACGT 11 12 APOE, exon3, Cys112Arg 336A, reverse, GCCGCTCACGT 11 13 APOE, exon3, Arg158Cys 474C, Forward, AGAAGCGCCTG 11 14 APOE, exon3, Arg158Cys 474T, Forward, AGAAGTGCCTG 11 15 APOE, exon3, Arg158Cys 474A, Forward, AGAAGAGCCTG 11 16 APOE, exon3, Arg158Cys 474G, forward, AGAAGGGCCTG 11 17 F5, exon10, Arg506Gln 1691G, Forward, CAGGCGAGGAA 11 18 F5, exon10, Arg506Gln 1691A, Forward, CAGGCAAGGAA 11 19 F5, exon10, Arg506Gln 1691C, forward, CAGGCCAGGAA 11 20 F5, exon10, Arg506Gln 1691T, Forward, CAGGCTAGGAA 11 21 F2, 3'-UTR, G20210A 20210G, Forward, TCAGCGAGCCT 11 22 F2, 3'-UTR, G20210A 20210A, Forward, TCAGCAAGCCT 11 23 F2, 3'-UTR, G20210A 20210C, Forward, TCAGCCAGCCT 11 24 F2, 3'-UTR, G20210A 20210T, Forward, TCAGCTAGCCT 11 25 FGB, 5'-UTR, G (-455) A -455G, forward, TTTAATGGCCCCT 13 26 FGB, 5'-UTR, G (-455) A -455A, forward, TTTAATAGCCCCT 13 27 FGB, 5'-UTR, G (-455) A -455C, forward, TTTAATCGCCCCT 13 28 FGB, 5'-UTR, G (-455) A -455T, forward, TTTAATTGCCCCT 13 29 GP1BA, 5'-UTR, T (-5) C -5T, forward, CACAGGTCCTCAT 13 30 GP1BA, 5'-UTR, T (-5) C -5C, forward, CACAGGCCCTCAT 13 31 GP1BA, 5'-UTR, T (-5) C -5A, forward, CACAGGACCTCAT 13 32 GP1BA, 5'-UTR, T (-5) C -5G, forward, CACAGGGCCTCAT 13 33 FABP2, exon2, Ala54Thr 244G, Forward, CAAGCGCTTTTCG 13 34 FABP2, exon2, Ala54Thr 244A, Forward, CAAGCACTTTTCG 13 35 FABP2, exon2, Ala54Thr 244C, Forward, CAAGCCCTTTTCG 13 36 FABP2, exon2, Ala54Thr 244T, Forward, CAAGCTCTTTTCG 13 37 PAI-1, 5'-UTR, 5G / 4G -675G, forward, ACACGTGGGGGAG 13 38 PAI-1, 5'-UTR, 5G / 4G -675, forward, ACACGTGGGGAG 12 39 PAI-1, 5'-UTR, 5G / 4G -675C, forward, ACACGTCGGGGAG 13 40 PAI-1, 5'-UTR, 5G / 4G -675A, forward, ACACGTAGGGGAG 13 41 PAI-1, 5'-UTR, 5G / 4G -675T, forward, ACACGTTGGGGAG 13 42 IL-6, 5'-UTR, G (-174) C -174G, forward, TCTTGCGATGCTA 13 43 IL-6, 5'-UTR, G (-174) C -174C, forward, TCTTGCCATGCTA 13 44 IL-6, 5'-UTR, G (-174) C -174A, forward, TCTTGCAATGCTA 13 45 IL-6, 5'-UTR, G (-174) C -174T, forward, TCTTGCTATGCTA 13 46 NOS3, exon7, Glu298Asp 894G, Forward, GATGAGCCCCCAG 13 47 NOS3, exon7, Glu298Asp 894T, Forward, GATGATCCCCCAG 13 48 NOS3, exon7, Glu298Asp 894A, Forward, GATGAACCCCCAG 13 49 NOS3, exon7, Glu298Asp 894C, Forward, GATGACCCCCCAG 13

< Example  5> Early diagnosis chip for stroke chip Production

Premature stroke chip provided in a preferred embodiment of the present invention MTHFR (5,10-methylenetetrahydrofolate reductase) C677T, AGT (Angiotensinogen) Met235Thr, APOE (Apolipoprotein E) ε2 / ε3 / ε4, F5 (Coagulation factor V Leiden) Arg506Gln, Coagulation factor II prothrombin (F2) G20210A, FGB (Fibrinogen) G (-455) A, GP1BA (Glycoprotein Ib, platelet alpha polypeptide) T (-5) C, FABP2 (Fatty acid-binding protein 2) Ala54Thr, PAI-1 (Plasminogen activator inhibitor-1) 5G / 4G, IL-6 (Interleukin-6) G (-174) C, Nitric oxide synthase 3 (NOS3) Glu298Asp, 49 probes integrated for SNP analysis of 11 genes It is. And the probe for human beta-globin is immobilized as an internal control. The chip layout was designed to include eight grids for the analysis of a total of 12 gene monobasic polymorphisms including an internal control on a single chip substrate, and an 8 well hybridization reaction chamber was used. It was designed to analyze the 8 individual samples at the same time (see Figure 2).

After thawing at 70 ° C., the probe stock was stored at room temperature, and then diluted with deionized sterile tertiary distilled water to a final concentration of 100 μM and used as a working stock. Each probe diluted 50-fold diluting working stock was subjected to a 1: 7-10 ratio (v / v) with 3X SSC spotting solution (500 mM NaCl, 3 mM sodium citrate, 1.5 MN, N, N-trimethylglycine, pH 6.8). By mixing, the concentration range of the probe dispensed into the final 96 well plate was 20-30 pmole / uL. The plate was mounted on a Microssys 5100 microarrayer (Cartesian Technologies, Ann Arbor, MI, USA) from which an aldehyde-, thioisocyanate-activated glass slide (CEL associates Inc., Houston, TX, USA) Two probes were spotted in order on the surface of the epoxy-activated plastic chip or gold film. The average diameter of the spots is 80-140 micrometers and the distance between spots was maintained at 350-500 micrometers to minimize cross-talk effects between spots. Chip fabrication was carried out in a class 10,000 room maintaining 75% humidity. The chip spotted with the probe was baked at 120 ° C. for 1 hour, and then washed for 3 minutes in 0.25% SDS (Sodium dodecyl sulfate) solution, and then again washed with sterile tertiary distilled water. The probe was then blocked by reacting the chip with a solution containing 0.2% sodium borohydride (NaBH ₄ ). After washing twice with 3 distilled water, the water was removed and stored in a desiccator (dessicator) until the point of use.

< Example  6> Early Stroke Diagnosis Chip chip ) Hybridization ( hybridization ) Response and result analysis

After performing a multiplex PCR reaction for early diagnosis of stroke according to a preferred embodiment of the present invention, 15 μL of the PCR reaction product and 25 uL of deionized tertiary distilled distilled water are added thereto, followed by heat denaturation at 95 ° C. for 5 minutes, followed by ice. The stomach was preserved for 5 minutes and spun down with a centrifuge. An 8 well hybridization chamber was placed on the chip surface and the well top was covered with a well cover. Thereafter, 60 uL of a hybridization reaction solution (3X SSC, 0.1% SDS, 0.2 mg / mL bovine serum albumin, pH 7) was preheated to the reaction mixture solution at 50 ° C. in the reaction mixture solution. After the addition and mixing, the reaction solution mixture was injected through the hole of the well cover, and care was taken not to generate bubbles. After fixing the chamber lid, hybridization reaction was performed at 58 ° C. for 30 minutes to induce specific nucleotide complementary binding between the probe on the chip surface and the multiplex PCR reaction product. The well cover of the chip surface where the hybridization reaction was completed was removed, the chip was immersed in the washing buffer 1 (0.1X SSC, 0.05% SDS) solution, washed with stirring at 2,000 rpm for 2 minutes, and repeated. After washing with washing solution 2 (2X SSC, 0.1% SDS) solution at 2,000 rpm for 2 minutes (washing) was repeated. Thereafter, the chips were dried by immersion in deionized tertiary sterile distilled water twice and centrifuged at 1,000 rpm. The chip was read using a ScanArray Lite (Packard Instrument Co., Meriden, CT, USA) scanner, and the mean fluorescence intensity and standard of the positive control spots using the analysis software (QuantArray 2.0). The signal-to-noise (S / R) ratio was obtained by comparing the error with the values around the spot, and when this value was 5 or more, it was scored as a positive value.

1 is a single nucleotide polymorphism (SNP) position, amino acid substitution occurrence of a total of 11 genes for stroke expression prediction analysis included in order to realize the purpose of early diagnosis of stroke according to the present invention, intracellularly related to stroke occurrence of the gene A table summarizing the mechanism of action and allelic frequency is shown.

Figure 2 is a schematic diagram of a chip for the early diagnosis of stroke produced according to the present invention. A chip grid in which probes of 11 stroke expression predictive assay genes were arranged was shown, and an 8-well hybridization reaction between the fluorescent DNA-labeled target DNA and the probes arranged on the chip was performed. The hybridization reaction chamber was schematically shown.

Figure 3 after the chip is a probe immobilized in accordance with the present invention, amplified target gene by multiplex-PCR reaction with the genomic DNA isolated from the sample as a template, hybridization with the selective probes on the chip (hybridization) Is a flow chart showing the main steps of the chip analysis process that analyzes the genetic predisposition for predicting stroke expression by performing the

4 is a chip scan image (left picture) and a chip grid (right picture) showing clinical analysis results of analytical target genes (five species) related to blood coagulation mechanisms in order to realize the purpose of early stroke diagnosis according to a preferred embodiment of the present invention. Picture). Analysis results of the MTHFR 677CT hetero genotype, F5 1691AA mutant genotype, F2 20210GA hetero genotype, FGB (-455) GA hetero genotype, GP1BA (-5) TT wild type genotype can be confirmed.

5 is a chip scan image (left picture) and a chip grid showing the results of clinical analysis of analytical target genes (five species) related to homeostasis of cerebrovascular vasculature in order to realize the purpose of early stroke diagnosis according to an exemplary embodiment of the present invention. (Right picture). Analysis results of AGT 704TC hetero genotype, APOE ε2 / ε4 genotype, FABP 244GA hetero genotype, NOS3 894TT mutant genotype can be confirmed.

Figure 6 shows a chip scan image (left picture) and a chip grid (right picture) showing clinical analysis results of a total of 11 different analysis target genes for realizing the purpose of early stroke diagnosis according to a preferred aspect of the present invention. MTHFR 677TT mutant genotype, AGT 704TC heterogenotype, APOE ε3 / ε4 genotype, F5 1691AA mutant genotype, F2 20210GA heterogenotype, FGB (-455) AA mutant genotype, GP1BA (-5) TC heterogenotype, FABP 244GG wild type genotype, PAI Analysis results of -1 (-675) ins genotype, IL-6 (-174) GG wild type genotype, and NOS3 894TT mutant genotype can be confirmed.

FIG. 7 is a schematic diagram illustrating a duplex structure formed between SNP sites of a target gene amplified through multiplex-PCR and probes immobilized on a surface for early diagnosis of stroke according to a preferred embodiment of the present invention. As an example, the analysis results of a GP1BA (-5) TT wild-type genotype specimen show that a probe specific for the wild-type GP1BA gene forms a stable duplex secondary structure with a target SNP region. However, cytosine, the SNP base of the GP1BA (-) 5T mutant probe, is located in the center of the base of the probe and is unable to form a stable duplex secondary structure that is mismatched with the complementary target SNP site base. After the reaction, the fluorescence signal is removed during the washing process. The SNP site is located at the central of the 11-13 base length probe, has no hairpin structure at the 3'-end, and preferably has a high specificity of SNP discrimination that does not include a dimer structure. Probes were designed.

<110> PARK, MinKoo <120> Stroke early diagnosing chip and screening analysis <160> 49 <170> KopatentIn 1.71 <210> 1 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for MTHFR 677C allele <400> 1 cgggagccga ttt 13 <210> 2 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for MTHFR 677T allele <400> 2 cgggagtcga ttt 13 <210> 3 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for MTHFR 677 <400> 3 cgggagacga ttt 13 <210> 4 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for MTHFR 677 <400> 4 cgggaggcga ttt 13 <210> 5 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for AGT 704T allele <400> 5 tgtccatggt g 11 <210> 6 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for AGT 704C allele <400> 6 tgtccacggt g 11 <210> 7 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for AGT 704 <400> 7 tgtccaaggt g 11 <210> 8 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for AGT 704 <400> 8 tgtccagggt g 11 <210> 9 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for APOE 336T allele <400> 9 gccgcacacg t 11 <210> 10 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for APOE 336C allele <400> 10 gccgcgcacg t 11 <210> 11 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for APOE 336 <400> 11 gccgcccacg t 11 <210> 12 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control for APOE 336 <400> 12 gccgctcacg t 11 <210> 13 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for APOE 474C allele <400> 13 agaagcgcct g 11 <210> 14 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for APOE 474T allele <400> 14 agaagtgcct g 11 <210> 15 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control for APOE 474 <400> 15 agaagagcct g 11 <210> 16 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for APOE 474 <400> 16 agaagggcct g 11 <210> 17 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for F5 1691G allele <400> 17 caggcgagga a 11 <210> 18 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for F5 1691A allele <400> 18 caggcaagga a 11 <210> 19 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for F5 1691 <400> 19 caggccagga a 11 <210> 20 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for F5 1691 <400> 20 caggctagga a 11 <210> 21 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for F2 20210G allele <400> 21 tcagcgagcc t 11 <210> 22 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> probe for F2 20210A allele <400> 22 tcagcaagcc t 11 <210> 23 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for F2 20210 <400> 23 tcagccagcc t 11 <210> 24 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for F2 20210 <400> 24 tcagctagcc t 11 <210> 25 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for FGB -455G allele <400> 25 tttaatggcc cct 13 <210> 26 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for FGB -455A allele <400> 26 tttaatagcc cct 13 <210> 27 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for FGB -455 <400> 27 tttaatcgcc cct 13 <210> 28 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for FGB -455 <400> 28 tttaattgcc cct 13 <210> 29 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for GP1BA -5T allele <400> 29 cacaggtcct cat 13 <210> 30 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for GP1BA -5C allele <400> 30 cacaggccct cat 13 <210> 31 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for GP1BA -5 <400> 31 cacaggacct cat 13 <210> 32 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for GP1BA -5 <400> 32 cacagggcct cat 13 <210> 33 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for FABP2 244G allele <400> 33 caagcgcttt tcg 13 <210> 34 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for FABP2 244A allele <400> 34 caagcacttt tcg 13 <210> 35 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for FABP2 244 <400> 35 caagcccttt tcg 13 <210> 36 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for FABP2 244 <400> 36 caagctcttt tcg 13 <210> 37 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for PAI-1 -675G allele <400> 37 acacgtgggg gag 13 <210> 38 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> probe for PAI-1 4G allele <400> 38 acacgtgggg ag 12 <210> 39 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for PAI-1 -675 <400> 39 acacgtcggg gag 13 <210> 40 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for PAI-1 -675 <400> 40 acacgtaggg gag 13 <210> 41 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for PAI-1 -675 <400> 41 acacgttggg gag 13 <210> 42 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for IL-6 -174G allele <400> 42 tcttgcgatg cta 13 <210> 43 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for IL-6 -174C allele <400> 43 tcttgccatg cta 13 <210> 44 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for IL-6 -174 <400> 44 tcttgcaatg cta 13 <210> 45 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for IL-6 -174 <400> 45 tcttgctatg cta 13 <210> 46 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for NOS3 894G allele <400> 46 gatgagcccc cag 13 <210> 47 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> probe for NOS3 894T allele <400> 47 gatgatcccc cag 13 <210> 48 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for NOS3 894 <400> 48 gatgaacccc cag 13 <210> 49 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> negative control probe for NOS3 894 <400> 49 gatgaccccc cag 13  

Claims (12)

Single-nucleotide polymorphism (SNP) high-throughput analysis of stroke marker predictive gene markers. The method of claim 1, further comprising: manufacturing a chip in which a probe modified with an amino modifier or a thiol modifier at the 5′- or 3′-end is spotted; Isolating genomic DNA from blood; Preparing a target DNA amplification product to include fluorescent dyes while amplifying probe target sites of stroke expression predictive marker genes through multiplex polymerase chain reaction (PCR); Performing a hybridization reaction between the multiplex PCR reaction product and a probe immobilized on a chip surface and washing the hybridization reaction; Analyzing the SNP results of stroke expression predictive markers by scanning the chip with a laser of a wavelength specific to the fluorescent dye and measuring the fluorescence intensity according to the hybridization reaction result; Analyzing and integrating the SNP results of the stroke expression predictive marker genes and the test results of four biochemical markers consisting of MMP-9, TIMP-1, hs-CRP, and homocysteine to derive a disease expression predisposition; And Stroke analysis method comprising all of the above analysis step. delete delete The method of claim 1, wherein the high-throughput analysis is a method for rapidly analyzing a large amount of SNP results of a stroke marker predictive gene by scanning a signal intensity of a fluorescent dye included in a reaction compound with a laser having an appropriate absorbance. Means, DNA chip, PNA chip, Luminex (Luminex), Pyrosequencing (Pyrosequencing) one of the selected method. The stroke of claim 1, wherein the stroke comprises one or more DNA probes selected from the group consisting of deoxyribonucleic acid oligonucleotides having nucleotide sequences of SEQ ID NOs: 1-49 for implementing high-throughput assays. DNA chip for analysis. The method according to claim 1, comprising at least one PNA probe selected from the group consisting of PNA oligonucleotides having a nucleotide sequence of SEQ ID NOs: 1 to 49 for implementing high-throughput assays PNA chip for stroke analysis. The process of claim 6 or 7, which is an aldehyde-, thioisocyanate-, or carboxyl-activated glass slide, or an epoxy-activated plastic. ) Or a chip for stroke analysis consisting of a chip made of a gold film material. 8. The probe of claim 6 or 7, wherein the probe designed for SNP analysis of stroke expression predictive gene markers has been modified to locate an amine or thiol functional group at the 5'- or 3'-terminus. , the carbon between the 6-terminal nucleotide sequence and the probe-9 _{(- (CH 2) 6-9 -} ) or thymidine (thymine) base, 12-15 (-d (T) _12-15 -) spacer (spacer) of Probe was added, and the length was selected within the range of 9-15 nucleotides. delete The multiplex-PCR kit of claim 1, wherein the multiplex-PCR kit amplifies one or more target DNA fragments selected from the probe sequence regions of SEQ ID NOs: 1 to 49 for SNP analysis of stroke expression predictive gene markers. delete

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