KR20030013019A - Diagnosis kit and diagnosis apparatus for multiplication disease of trinucleotide repeated sequence - Google Patents

Abstract

PURPOSE: Provided are a diagnosis kit and a diagnosis apparatus for multiplication disease of trinucleotide repeated sequence, thereby simply and accurately diagnosing a patient suffering from multiplication disease of trinucleotide repeated sequence with a small amount of a test sample. CONSTITUTION: The diagnosis kit for multiplication disease of trinucleotide repeated sequence comprises the PCR primer which amplifies a specific trinucleotide repeated sequence in DNA collected from a test sample; a PCR buffer solution and enzymes; and a transfer and separation unit which transfers and separates PCR products according to a size by capillary electrophoresis.

Description

Diagnosis kit and diagnosis apparatus for multiplication disease of trinucleotide repeated sequence}

The present invention relates to a diagnostic kit and diagnostic device for trinucleic acid repeat sequence increase disease, and more particularly, to use a simple and accurate polymerase chain reaction (PCR), and to easily use for the diagnosis of patients and carriers as well as general screening tests. The present invention relates to a diagnostic kit and a diagnostic device for increasing trinucleic acid repeat sequence diseases.

In most cases, the causes and mechanisms of neurological diseases are not known exactly. However, some diseases have recently been found to be a direct cause of genes, and researches are being actively conducted.

In particular, since the (CGG) n repeat sequence was reported to increase in Fragile X syndrom patients in 1991, more than 13 neurological diseases have been known as trinucleic acid repeat sequence increasing diseases so far.

Trinucleic acid repeat sequence increase disease is a genetic disease caused by increasing nucleotide sequences repeated in trinucleic acid units such as CAG, GAA, CGG, etc., present in genomic DNA. In some cases, it has been reported that the number of patients increased from several times to several hundred times and in some cases thousands of times as compared to normal people.

These diseases can be broadly divided into two groups, the first group having an increased (CAG) n sequence in the coding sequence, having 10-30 repeats in a normal person, Some cases have 40-200 recurrences, such as Huntington's disease, Kennedy's disease, Spinocerebellar ataxia type 1,2,3,6,7, Dentetoru Bural Palidoruian atrophy (Dentato-rubral-pallido-luysian atrophy, DRPLA).

On the other hand, the second group of diseases has various repeat sequences such as CGG, CTG, GAA, and CCG, and the range of increase is much larger from several hundred to several thousand times, and is affected by the mother's system. Fraxile X Syndrom ), Myotonic dystrophy, Friedreich ataxia, and spinal cerebellar ataxia type8.

Table 1 shows the gene region and repeat sequence of the disease caused by the increased trinucleic acid repeat sequence, the range of normal and patients.

Disease name Gene region Repeat sequence Normal range Patient range Huntington's Disease (HD) 4p16.3 CAG 6-35 36-> 100 Kennedy Disease (SBMA) Xq21 CAG 9-35 38 (40) -62 Spinal cerebellar ataxia 1 (SCA1) 6p23 CAG 6-38 (39) 39 (41) -83 Spinal cerebellar ataxia 2 (SCA2) 12q24 CAG 14-31 32 (35) -77 Spinal cerebellar ataxia 3 (SCA3, MJD) 14q32.1 CAG 12-39 62-86 Spinal cerebellar ataxia 6 (SCA6) 19p13 CAG 4-17 21-30 Spinal cerebellar ataxia 7 (SCA7) 3p12-p21.1 CAG 7-35 37-200 DRPLA 12p CAG 3-35 (25) 49-88 Fraction X Syndrome (FRAXA) Xq27.3 CGG 6-54 200 (230)-> 2000 Fraser X-Site E (FRAXE) Xq28 CCG 6-25 > 200 Myotonic dystrophy (DM) 19q13 CTG 5-35 50-4000 Friedrich's ataxia (FA) 9q13-q21.1 GAA 7-22 200-1700 Spinal Cerebellar Ataxia 8 13q21 CTG 16-37 110-> 500

Although all of these diseases are very rare, some of the diseases are dominant, so if a mutation occurs in the gene, 50% of the next generation may be affected. In addition, some diseases, such as Huntington's disease and dystonia, are known at the time of onset, and only after 2 years of age are born, it is important to be diagnosed before symptoms appear.

Since almost 100% of these diseases are caused by a single mechanism of increase in trinucleic acid repeat sequence, molecular genetic diagnosis using them is very effective and necessary for confirming or detecting patients. In addition, the degree of trinucleic acid repeat sequence increase is closely related to the age of onset and the severity of the disease, and it is known that the risk increases with age. In carriers, the trinucleic acid repeat sequence is normal between the patient and the patient, thus having no symptoms but affecting the next generation. Therefore, precisely investigating the extent of trinucleic acid repeat sequence can surely diagnose the diseases and predict the prognosis.

In the past, a special chromosome test using blood of a patient was performed, but the diagnosis using the cytogenetic method inherent the possibility of false negative and false positive, and in fact, the diagnosis of the carrier was impossible.

Since it has been found that the increase in trinucleic acid repeat sequence causes these diseases, it is very efficient and accurate to diagnose the disease by predicting the length of trinucleic acid repeat sequence using molecular genetic method.

Southern blotting is a very accurate diagnosis, but it is time consuming, expensive, complex and inadequate for general screening.

The most commonly used PCR can be used for diagnosis by amplifying the trinucleic acid repeat sequence region.However, it is difficult to determine the exact size of the PCR amplification product on the agarose gel. The problem is that screening carriers is impossible.

Therefore, there is a need for a new method that can be easily used for diagnosis of patients and carriers as well as general screening tests using simple and accurate PCR methods.

The present invention has been made to solve the problems of the prior art, the object of the present invention is to increase the trinucleic acid repeat sequence that can be easily used for the diagnosis of patients and carriers as well as general screening test using a simple and accurate PCR method To provide a diagnostic kit of the disease.

It is still another object of the present invention to provide a diagnostic apparatus for trinucleic acid repeat sequence increase disease that can be easily used for diagnosis of patients and carriers as well as general screening tests using a simple and accurate PCR method.

Figure 1a is a diagram showing the SCA1, SCA3, SCA6, SCA7, SCA8 PCR results of a normal sample,

Figure 1b is a diagram showing the PCR results of SBMA patients,

Figure 1c is a diagram showing the PCR results of HD patients,

Figure 1d is a diagram showing the PCR results of the patients with Fractions X syndrome,

Figure 1e is a diagram showing the PCR results of DRPLA, DM, FRAXE patients,

Figure 2a is a diagram showing the results of the Fractional X syndrome PCR of a normal sample,

FIG. 2B is a view for explaining the sample type, size (bp), and repetition of each lane of FIG. 2A;

Figure 3a is a diagram showing the SCA3, SCA6, SCA7, SCA8 PCR results of the normal sample,

3B is a view for explaining the sample type, size (bp) and repetition of each lane of FIG. 3A;

Figure 4a is a diagram showing the DM and DRPLA PCR results,

4B is a view for explaining the sample type, size (bp) and repetition of each lane of FIG. 4A.

Figure 5a is a diagram showing the SBMA PCR results of normal people and patients,

5B is a view for explaining the sample type, size (bp) and repetition of each lane of FIG. 5A;

Figure 6a is a diagram showing the PCR results of the Huntington's disease patients,

FIG. 6B is a diagram for explaining sample type, size (bp), and repetition of each lane of FIG. 6A;

7 is a view showing the results of SBMA PCR and DOP-PCR using DNA isolated from the mother's blood,

8 is a view showing the overall shape of a microCE chip according to the present invention,

9 is a diagram illustrating a diagnosis apparatus using a microCE chip according to the present invention.

In order to achieve the above object, the present invention provides a diagnostic kit for trinucleic acid repeat sequence increase disease according to the present invention, a primer for amplifying a specific trinucleic acid repeat sequence region from DNA collected from a sample; Polymerase chain reaction buffer and enzyme; It includes a mobile separation means that can be separated by moving the amplification product by the polymerase chain reaction according to the size by micro capillary electrophoresis method.

According to the above configuration, the number of specific trinucleic acid repeat sequences amplified by PCR can be easily and accurately grasped by using microcapillary electrophoresis, and thus there is an advantage that it can be easily used for diagnosis of patients and carriers as well as general screening tests. .

According to a second aspect of the present invention, in the diagnostic kit for trinucleic acid repeat sequence increase disease according to the present invention, the primer comprises an oligonucleotide having one or more nucleotide sequences selected from SEQ ID NO: 1 to SEQ ID NO: 26. do.

According to the above configuration, it is possible to efficiently amplify a specific repeat sequence of the trinucleic acid repeat sequence increase disease, Huntington's disease, Kennedy's disease (SBMA), spinal cerebellar ataxia 1 (SCA1), spinal cerebellar ataxia 2 (SCA2) , Spinal Cerebellar Ataxia 3 (SCA3), Spinal Cerebellar Ataxia 6 (SCA6), Spinal Cerebellar Ataxia 7 (SCA7), DRPLA, Fractional X Syndrome (FRAXA), Fractional Esite (FRAXE), Myotonia There is an advantage that more precise diagnosis of trinucleic acid repeat sequence disorders of sexual dystrophy (DM) and Friedreich's ataxia.

According to a third aspect of the present invention, the mobile separation means in the diagnostic kit for trinucleic acid repeat sequence disorders, the microcapillary electrophoretic chip is formed on the glass substrate, a micrometer channel and the container; And a movement control means capable of controlling the movement of the trinucleic acid repeat sequence in the microcapillary electrophoretic chip by an electric field.

According to the above structure, since several samples can be analyzed on the chip of several cm size, it can contribute to the miniaturization of a diagnostic apparatus, and reduction of manufacturing cost. In addition, the concept of a lab-on-a-chip that performs PCR, separation, and detection on a single chip has a great possibility of automating a diagnostic apparatus. In addition, since only a small amount of the sample can be diagnosed, there is an advantage that the fetus can be diagnosed correctly as a prenatal diagnosis using amniotic fluid or fetal cells separated from maternal blood.

According to the fourth aspect of the present invention, a diagnostic device for trinucleic acid repeat sequence increase disease according to the present invention comprises: a primer for amplifying a specific trinucleic acid repeat sequence region from DNA collected from a sample; Polymerase chain reaction buffer and enzyme; A diagnostic kit comprising a mobile separation means capable of moving separation of the amplified product by the polymerase chain reaction according to the size by micro capillary electrophoresis; Detecting means for detecting a moving position of the trinucleic acid repeat sequence separated by the moving separating means; And analyzing means for analyzing the number of trinucleic acid repeat sequences based on the results detected by the detecting means.

According to the above configuration, it is possible to provide a small and inexpensive automated diagnostic device for trinucleic acid repeat sequence disorders that can accurately and quickly diagnose trinucleic acid repeat sequence disorders with a small amount of samples.

According to a fifth aspect of the present invention, a method for diagnosing a trinucleic acid repeat sequence increase disease includes amplifying a specific trinucleic acid repeat sequence portion of a DNA of a sample by a polymerase chain reaction; Separating the amplified product amplified by the polymerase chain reaction from a predetermined sample by micro capillary electrophoresis; And analyzing the number of trinucleic acid repeat sequences by detecting a moving position of the separated DNA.

According to the method, even a small bp difference can be accurately distinguished, and there is an advantage that a small amount of sample can be easily used for diagnosis of patients and carriers as well as general screening tests.

The present invention is called a micro capillary electrophoresis chip (micro CE chip) with an inner diameter of about 10 μm to 100 μm with PCR in order to accurately and simply diagnose trinucleic acid repeat sequence increase diseases using a small amount of samples. Was used).

The microCE chip is a method of analyzing DNA by applying a high voltage to the channel using a capillary tube of several μm made by dry or wet etching, which is a method used for semiconductor processing on a glass substrate. In other words, since DNA has a negative charge property, when an electric field is applied, a flow toward the anode occurs. At this time, the DNA has a velocity distribution due to its size and charge ratio, and is a technique of separating DNA using such a speed difference. Therefore, the present invention is focused on the fact that the trinucleic acid repeat disease is a disease determined by the number of repeating base pairs of each DNA, so that if the DNA chain length can be quantitatively determined, it can be determined whether the disease is present. It is.

On-chip analysis system using the capillary electrophoresis as described above is relatively easy to manufacture because it can control the fluid flow in the microchip by the electric field alone without the fluid control devices such as pumps and valves, As a result, the analysis period is shorter and the separation efficiency is increased.

CE is also a rapidly evolving separation technology that can control selectivity using a variety of separation modes and with the potential for small sample consumption, on-capillary detection, quantitative analysis and automation.

Therefore, the present invention, which uses this technique to test for genetically modified diseases such as trinuclear repeat disease, can significantly reduce the amount of sample and analysis time, as well as the accuracy, reproducibility, and simplicity of experiment, compared to the conventional method. It also has an incomparable advantage. In addition, the ability to analyze multiple samples on a chip of several centimeters in size makes the device compact and cost competitive. In addition, the diagnostic kit according to the present invention has a high possibility of automation by introducing a lab-on-a-chip concept that performs PCR, separation, and detection on a single chip.

It can be analyzed with only a small amount of sample, and the method is simple, and the resolution is very high, so that the difference of small bp can be accurately distinguished, which is a breakthrough method for diagnosing trinucleic acid repeat sequence increased diseases.

Hereinafter, the present invention will be described in detail by the following examples. The following examples are provided to illustrate the present invention, and the scope of the present invention should not be construed as being limited by the following examples.

Example

1) material

The patient's DNA was provided by Seoul National University Hospital and the Department of Genetic Diseases, National Institute of Health. The rest of the samples were used after extracting DNA from whole blood of normal persons using QIAamp DNA mini kit (QIAGEN, USA). For prenatal diagnosis, the mother's blood was collected at 16-18 weeks of gestation, and nucleated red blood cells (single nucleated RBCs (nRBCs)) were isolated to extract fetal DNA. Specifically, after dissolving in 5 μl of isolated nucleated red blood cells and soaking in liquid nitrogen three times for 5 seconds, 5 μl of lysis buffer (lysis buffer, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 500 μg / ml Proteinase) K) is added to react at 60 ° C for 2 hours and the enzyme is inactivated at 94 ° C for 2 minutes.

2) primer

Table 2 shows the sequences of the primers according to the invention. Each primer was used in synthetic order from Bionic Inc., Canada. For other reagents, a special reagent was used.

Disease name PRIMER order HD HDF 5 'ATG AAG GCC TTC GAG TCC CTC 3' HDR 5 'CAG CAG CGG CTG TGC CTG 3' SBMA SBF 5 'TCC AGA ATC TGT TCC AGA GCG 3' SBR 5 'GTG AAG GTT GCT GTT CCT CAT 3' SCA1 S1F 5 'CCA GCG CTC CCA GCT GGA 3 S1R 5 'TGG TTC TGC TGG GCT GGT G 3 SCA2 S2F 5 'GCC CGG CGT GCG AGC CGG TGT ATG 3' S2H 5 'CTT GCG GAC ATT GGC AGC CGC GGG 3' SCA3 53F 5 'GTC TAG .ATT TCC TAA GAT CAG CAC 3' S3R 5 'AAG TGC TCC TGA ACT GGT GGC TGG 3' SCA8 S6F 5 'CCT ATT CCC CTG TGA TCC GTA AGG 3' S6R 5 'CTG GGC GAG CGG CCG CTG CTG TGG 3' SCA7 S7F 5 'GTT ACA TTG TAG GAG CGG A 3' S7R 5 'ACG ACT GTC CCA GCA TCA.CTT C 3' ORPLA DRF 5 'ATG GCC GCC TCT TAG CCA ACA GCA 3' DRR 5 'CAT GGC GTA AGG GTG TGC GTG GTG 3' FRAXA XFl 5 'GCT CAG CTC CGT TTC GGT TTC ACT 3' XR1 5 'GCA CTT CCA CCA CCA GCT CCT CCA 3' FRAXE X2F 5 'GTG CTG CAC AGC CTG CGC CTT AC 3' X2R 5 'ATG CGC GGC CCT CGT ACG GAA G 3' DM MF 5 'CAG CTC CAG TCC TGT GAT C 3' MR 5 'TGG AGG ATG GAA CAC GGA C 3' FA FF 5 'CGG AGT TGA AGA CTA ACC TGG 3' FR 5 'TGG AGT AGC TGG GAT TAC AGG 3' SCA8 S8F 5 'GCA GTA TGA GGA AGT ATG G 3' S8H 5 'CTG TTG GCT GAA GCC CTA TC 3'

* SEQ ID NOs: 1 to 26 are given in the order of Table 2 above

As shown in Table 2, the primer for each disease is designed to selectively amplify only the repeat sequence of the gene region of the disease.

3) DOP-PCR

In case of using DNA isolated from fetal nucleated red blood cells, the whole genome is amplified by DOP-PCR before polymerase chain reaction. The primer sequence is 5 'CCG ACTCGA GNN NNN NAT GTG G 3' (SEQ ID NO: 27) and amplified using one primer.

The reaction consists of two steps. The PCR reaction solution composition used in each step is as follows.

Total amplification step (Total 10µl) 10X buffer (100 mM Tris-HCl, pH 8.3,50 mM KCl, 25 mM MgCl ₂ ) 1 μl 2.5mM dNTP mix 0.4 μl 100pmol primer 0.1 μl Taq DNA Polymerase 0.1U sample 5 μl Secondary Amplification Step (Total 50µl) 10X buffer (100mM Tris-HCl, pH 8.3,50mM KCl, 10mM MgCl ₂ ) 5 μl 2.5mM dNTP mix 2 μl 100pmol primer 0.1 μl Taq DNA Polymerase 0.1U Preamplification products 10 μl

4) Polymerase Chain Reaction (PCR)

PCR was performed using 75mM Tris-HCl (pH 9.0), 15mM KCl, 25mM MgCl ₂ , 0.1mg / ml BSA, 50µm dNTPs, 2X Band Doctor (Band Doctor, Solgent, Korea), 50pmol primer, 1 unit of Taq polymer Lase, 0.1 μg of DNA or DOP-PCR finished amplification products were added to react at a final volume of 25 μl.

Table 4 shows the PCR reaction solution composition.

PCR reaction solution composition Reaction condition (μl) 10X PCR Buffer 2.5 5X Band Doctor 10 2.5mM dNTPs One Primer 1 (F) One Primer 2 (R) One Taq polymerase 0.2 Template DNA One

PCR reactions were carried out 35 times under the following conditions.

-Total denaturation: 95 ℃, 2 minutes

-Denaturation: 95 ℃, 30 seconds

Primer binding: 30 seconds at different temperatures for different diseases (see Table 4)

Polymerization: 72 ° C, 30 seconds

-Last extension: 72 ℃, 3 minutes

PCR products produced through the above process was confirmed by electrophoresis on 2% agarose gel. The results are shown in Figs. 1A to 1E. Figure 1a is a SCA1, SCA3, SCA6, SCA7, SCA8 PCR PCR results of a normal sample (L is 100bp ladder, lanes 1-3 are SCA1, lanes 4-6 are SCA3, lanes 7-9 are SCA6, lanes 10-12 SCA7, lanes 13 to 15 are SCA8, lanes 3, 6, 9, 12, and 15 are negative controls, respectively, and FIG. 1B is a result of PCR of SBMA patients (L is 100bp ladder and lanes 1 to 13 are patient samples). , N denotes a negative control group, Figure 1c shows the results of PCR of HD patients (L is 100bp ladder, lanes 1 to 9 patient samples, N is a negative control group).

In addition, Figure 1d is a diagram showing the PCR results of patients with Fraction X syndrome (L is 100bp ladder, lane 1 is negative control group, lanes 2 to 20 are normal samples), Figure 1e is a PCR result of DRPLA, DM, FRAXE patients (L is 100bp ladder, lanes 1-4 are DRPLA, lanes 6-10 are DM, lanes 12 and 13 are FRAXE, and lanes 5, 11 and 14 are negative controls, respectively).

On the other hand, Table 5 below shows the primer binding temperature in the PCR reaction for each disease, and the length of the PCR product in the absence of repetition.

Disease name Primer binding temperature Length of PCR Amplification Products HD 64 ℃ 110 bp SBMA 58 ℃ 222 bp SCA1 57 ℃ 143bp SCA2 68 ℃ 80 bp SCA3 57 ℃ 278 bp SCA6 55 ℃ 165 bp SCA7 57 ℃ 270 bp DRPLA 60 ℃ 209 bp FRAXA 68 ℃ 220bp FRAXE 60 ℃ 151 bp DM 57 ℃ 216 bp FA 57 ℃ 157bp SCA8 55 ℃ 190 bp

5) DNA sequence analysis of amplified products

In order to verify the experiment, the nucleotide sequence of the PCR amplification product was analyzed using an automatic sequencer. After PCR, the amplified product was purified using a QIAquick PCR purification kit (QIAGEN, USA), and the reaction solution using an ABI PRISM ^TM Dye Terminator Cycle Sequencing kit. PCR sequencing was performed by adding primers used in PCR reaction with purified DNA at a concentration of 10-25 ng / μl per 20 μl. PCR sequencing was performed using GeneAmp PCR system 9600 (model 9600 Perkin-Elmer, Foster city, CA, USA) for 30 seconds at 96 ° C, 15 seconds at 50 ° C, and 4 minutes at 60 ° C. The reaction was performed 25 times in total. Unreacted nucleotides were removed using Centri-Sep ^™ spin columns Princeton Separations, Philadelphia, NJ, USA, and vacuum dried in a speed bag (Speed Vac, Savane Instruments, Farmingdate, NY, USA). 6 μl of loading buffer (5 μl of loading buffer deionized formamide, 1 μl of 25 mg EDTA (pH 8.0) containing 50 mg / ml blue dextran) was dissolved. After 2 minutes at 90 ℃ denatured just prior to loading electrophoresed ssikeul 1.5㎕ in denatured polyacrylamide gels of 4.75% to 15KW and ABS children analyzing the nucleotide sequence results in the prism 310 Genetic analyst riser (ABI PRISM ^TM 310 Genetic analyzer) It was. The size of the base sequence resulting from the analysis is shown in Figs. 3b, 4b, 5b and 6b. As shown in the figures, the difference between the sequence analysis result and the result by the analysis method using the microCE chip is only 1-2 repetition, it can be seen that the same in the patient or carrier diagnosis.

6) Analysis using micro CE chip

8 shows a schematic view of a microCE chip. As shown in FIG. 8, the microCE chip is composed of a substantially cross-shaped channel on the chip, and a container capable of holding a sample and a buffer solution formed at each end of the channel. In FIG. 8, the sample injection container 10 is formed on the right side in the lateral direction, and the sample discharge container 12 is formed on the left side. In addition, a buffer solution injection container 14 containing a buffer solution is formed in the longitudinal upper portion of FIG. 8, and a buffer solution discharge container 16 is formed in a lower part thereof. The channel between the sample injection container 10 and the sample discharge container 12 is an injection channel 22, and the channel between the buffer injection container 14 and the buffer solution discharge container 16 is a separation channel 24. The lower portion of the separation channel 24 is a detector 26.

PCR amplification products identified by electrophoresis on 2% agarose gels were analyzed using an Agilent 2100 Bioanalyzer, USA. DNA 500 LabChip, which can be measured with 5% resolution accuracy in the range of DNA 25-500bp, was used and kept constant at 30 ° C at the experiment temperature.

First, mix the gel and dye in the unit amount supplied from Agilent, put it in the chip, and add 1 μl DNA 500 ladder to the ladder hole. The dye is attached to the DNA as it moves on the micro CE chip, so that the DNA movement position can be detected by a laser detector. After that, 1 μl of DNA to be analyzed and 5 μl of size marker (15/600 bp) were put into each separation hole. Finally, the chip was returned to the vortex for 1 minute and then placed in an Agilent 2100 bioanalyzer.

The results are shown in FIGS. 2 to 7. Figure 2a shows the results of the Fractional X syndrome PCR of the normal sample, Figure 3a shows the SCA3, SCA6, SCA7, SCA8 PCR results of the normal sample, Figure 4a shows the DM and DRPLA PCR results, Figure 5a shows the SBMA PCR of the normal and the patient 6A shows the PCR results of samples of Huntington's disease patients, respectively. 2-6 is a figure for demonstrating the sample type, size (bp), and repetition of each lane of each a figure.

Figure 7 shows the SBMA PCR and DOP-PCR results using DNA isolated from the mother's blood. In Figure 7, M is a marker of 100bp size, lane 1 shows the SBMA PCR results of DOP-PCR amplification products, lane 2 shows the DOP-PCR results of DNA isolated from the mother's blood. DOP-PCR amplification product amplified the whole genome appears on the gel smear (smear), the product size of lane 1 used for this SBMA PCR corresponds to 17 iterations of 272bp.

7) Manufacture of diagnostic device using micro CE chip

Referring to Figure 8 will be described a method of manufacturing a microCE chip used in the present invention. A glass wafer (0.5mm thick Schott Borofloat) was used, and the width of the channel was 70 μm, depth 15 μm, and the separation channel 38 mm. After the Cr / Au mask was placed on the wafer, the positive photoresist (AZ 1512) was spin-coated at 500 rpm for 5 seconds and 4000 rpm for 30 seconds. The wafer on which the photoresist (PR) was loaded was soft baked at 90 ° C. for 30 minutes and subjected to UV exposure with a Cr photomask to etch the pattern on the wafer. The exposure time at this time was 10 seconds. After the photoresist development, hard bake was conducted at 115 ° C. for 30 minutes. After hard baking, Au was removed in tens of seconds using aqua regia. Cr was stripped off using a Cr7 etchant. The channel was then etched using buffer HF solution. Cover glass (0.5mm thick Schott Borofloat) with a 15mm hole in each reservoir location was made to thermally bond with the channeled glass.

The gel used was added acrylamide to 45 mM Tris / 45 mM borate / 1 mM EDTA / 8.3 M urea (pH 8.3) and placed in the channel, followed by adding 2.5 μl of 10% ammonium persulfate and TEMED. The polymerization was carried out.

The electrical separation and detection of the sample is done using a MP5P24 power supply made by Spellman, USA, and a laser detector made by the laboratory. Voltage regulation is available with LabVIEW (National Instruments, USA). 9 is a schematic diagram of a diagnostic device for trinucleic acid repeat sequence increase disease according to the present invention.

Hereinafter, the DNA separation mechanism will be briefly described.

Before sample injection, high voltage is applied to the sample container and the sample discharge container is grounded to move the sample, but the high voltage is also applied to the buffer container and the buffer discharge container to prevent the sample from flowing into the separation channel. Adjust to move only. When the sample is injected, the voltage is applied to the injection channel at 170V / cm for 60 seconds and the voltage is cut off momentarily, and the separation process is performed by applying a voltage of 250V / cm to the separation channel. That is, if the sample in the sample injection part flows toward the separation channel by changing the voltage configuration, and the sample injection and separation occurs, the sample plug can be injected with high precision by minimizing the space spread of the sample plug. have.

When the high voltage is applied to the channel as described above, the DNA having negative charge property moves in the direction of the anode, and the DNA is separated by the velocity distribution due to the size and charge ratio of the DNA.

As described above, according to the present invention, amplification products can be efficiently obtained by selectively amplifying the trinucleic acid repeat sequence region of each disease by using the newly prepared primer.

These amplification products can be analyzed using a microcapillary electrophoresis chip in a short time to the exact length of the trinucleic acid repeat sequence. Therefore, early diagnosis is possible before the confirmation or signs of patients with similar symptoms.

In addition, the analysis according to the present invention is simple and inexpensive as compared to the existing method, and can be used for family history research because there is no problem to use it as a general screening test.

In addition, since only a small amount of the sample can be diagnosed, fetal disease can be accurately diagnosed as a prenatal diagnosis using amniotic fluid or fetal cells separated from maternal blood.

Claims (23)

A primer for amplifying a specific trinucleic acid repeat sequence region from DNA collected from a sample; Buffer and enzyme for polymerase chain reaction; Diagnosis kit of trinucleic acid repeat sequence increase disease comprising a mobile separation means for moving separation of the amplification product by polymerase chain reaction according to the size by micro capillary electrophoresis. According to claim 1, wherein the primer is a diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that it comprises an oligonucleotide having at least one base sequence selected from SEQ ID NO: 1 to SEQ ID NO: 26. The disease according to claim 1 or 2, wherein the disease is Huntington's disease, Kennedy's disease (SBMA), spinal cord cerebellar ataxia 1 (SCA1), spinal cord cerebellar ataxia 2 (SCA2), spinal cord Cerebellar Ataxia 3 (SCA3), Spinal Cerebellar Ataxia 6 (SCA6), Spinal Cerebellar Ataxia 7 (SCA7), DRPLA, Fraction X Syndrome (FRAXA), Fractional Esite (FRAXE), Myotonic dystrophy (DM), diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that at least one selected from Friedrich ataxia. According to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that to diagnose Huntington's disease using the same. According to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4, using the same diagnostic kit for trinucleic acid repeat sequence increase disease, characterized in that to diagnose Kennedy disease. According to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6, diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that for using this to diagnose SCA1. According to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8, diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that for using this to diagnose SCA2. According to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10, diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that for using this to diagnose SCA3. According to claim 2, wherein the primers are oligonucleotide having a nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12, and diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that using this to diagnose SCA6. According to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14, diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that for using this to diagnose SCA7. According to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 16, diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that for diagnosing DRPLA using it. The diagnostic kit according to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 18, and the FRAXA is diagnosed using the primer. According to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 19 and SEQ ID NO: 20, diagnostic kit of trinucleic acid repeat sequence increase disease, characterized in that for using the diagnosis of FRAXE. The diagnostic kit according to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 21 and SEQ ID NO: 22, and the DM is diagnosed using the primer. The diagnostic kit according to claim 2, wherein the primer is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 23 and SEQ ID NO: 24, and the FA is diagnosed using the primer. According to claim 2, wherein the primers are oligonucleotides having a nucleotide sequence of SEQ ID NO: 25 and SEQ ID NO: 26, using the diagnostic kit for trinucleic acid repeat sequence amplification disease, characterized in that the diagnosis of Friedreich ataxia. The method of claim 1, wherein the moving separating means, A microcapillary electrophoretic chip having a channel and a container formed in μm on a glass, plastic or silicon substrate; And And a movement control means for controlling the movement of the trinucleotide repeat sequence in the microcapillary electrophoresis chip by an electric field. 18. The diagnostic kit according to claim 17, further comprising a fluorescent substance attached to the trinucleic acid repeat sequence separated by the mobile separation means and indicating a moving position. The diagnostic kit of trinucleic acid repeat sequence increase disease according to claim 1 or 2, wherein the sample is nucleated red blood cells isolated from whole blood or amniotic fluid or mother's blood. A primer for amplifying a specific trinucleic acid repeat sequence region from DNA collected from a sample; Polymerase chain reaction buffer and enzyme; A diagnostic kit comprising a mobile separation means for moving separation of the amplified product by the polymerase chain reaction according to the size by micro capillary electrophoresis; Detecting means for detecting a moving position of the trinucleic acid repeat sequence separated by the moving separating means; And And diagnostic means for analyzing the number of trinucleic acid repeat sequences based on the results detected by the detecting means. 21. The diagnostic device for trinucleic acid repeat sequence increase disease according to claim 20, wherein the detection means is an optical or electrochemical detection device for detecting a fluorescent substance attached to DNA. 22. The trinucleic acid repeat sequence according to claim 20 or 21, wherein the analysis means compares the number of trinucleic acid repeat sequences detected by the detection means with the number of trinucleic acid repeat sequences stored in the trinucleic acid repeat sequence disease. Diagnostic device for trinucleic acid repeat sequence increase disease, including a computer for analyzing the disease. Amplifying a specific trinucleic acid repeat sequence portion of DNA of the sample by a polymerase chain reaction; Moving and separating the amplified product amplified by the polymerase chain reaction from a predetermined sample by micro capillary electrophoresis; And Detecting a moving position of the separated DNA, and analyzing the number of trinucleic acid repeat sequences.