WO2003014396A1

WO2003014396A1 - Diagnosis method of multiplication disease of trinucleotide repeated sequence and a diagnosis kit

Info

Publication number: WO2003014396A1
Application number: PCT/KR2002/001489
Authority: WO
Inventors: Jeong-Mi Kim; Young-Eun Lee; Seung-Jae Baik; Hyun-Jung Kim; Sang-Yup Han
Original assignee: Biomedlab Corporation
Priority date: 2001-08-06
Filing date: 2002-08-06
Publication date: 2003-02-20
Also published as: KR20030013019A

Abstract

The present invention relates to a diagnosis method for a multiplication disease of repeated trinucleotide sequences and a diagnosis kit, and more particularly to a method for diagnosing Huntington disease, Kennedy disease, Spinocerabellar ataxia type 1, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Dentatorubral-pallidoluysian atrophy, Fragile X syndrome, Fragile E site, Myotonic dystrophy, Friedreich ataxia or Spinocerebellar ataxia type 8 by micro capillary electrophoresis,and thereby patients and silent carriers having a multiplication disease of repeated trinucleotide sequences can be easily detected.

Description

DIAGNOSIS METHOD OF MULTIPLICATION DISEASE OF TRINUCLEOTIDE REPEATED SEQUENCE AND A DIAGNOSIS KIT

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a method for diagnosis of a multiplication disease of repeated trinucleotide sequences, and a diagnosis kit for the same, and particularly, to a method for diagnosis of a multiplication disease of repeated trinucleotide sequences comprising the steps of amplifying the repeated trinucleotide sequence region by a polymerase chain reaction; analyzing the amplified product with micro capillary electrophoresis; and detecting the multiplied level of the repeated trinucleotide sequence, and to a diagnosis kit for the same.

(b) Description of the Related Art Multiplication diseases of repeated trinucleotide sequences are denoted as genetically inherited diseases in which sequence of three nucleotides is expanded or amplified in genomic DNA by several times through several hundreds times, and in certain cases, by several thousands times. So far, it has been found that the repeated trinucleotide sequences are expanded or amplified in 13 or more neurogenic diseases, since it was firstly reported that a (CGG)n repeated sequence was expanded and amplified in subjects suffering from Fragile X syndrome in 1991.

The multiplication diseases of repeated trinucleotide sequences may be classified into two groups. The first group will be designated as diseases characterized in having high numbers of (CAG)n repeated sequences (n = 40-200) in a coding sequence. For reference, a healthy individual has relatively low numbers of (CAG)n repeated sequences (n = 10-30). The multiplication diseases involved in the first classification of high (CAG)n repeats are exemplified as follows: Huntington's disease, Kennedy's disease, Spinocerebellar ataxia, and Dentatorubral pallidoluysian atrophy.

On the other hand, the second group will be designated as diseases characterized by having various nucleotide repeats such as CGG, CTG, GAA, and CCG that are expanded and amplified at a remarkably high level, such as several hundred times through several thousand times those of normal levels of a healthy individual. The multiplication diseases belonging to the second group may include, for example, Fragile X syndrome, Myotonic dystrophy, Friedrich's ataxia, and Spinocerebellar ataxia type 8. The following Table 1 shows the genetic region, the repeated sequence, and the multiplication level of healthy and affected subjects according to the multiplication diseases of repeated trinucleotide sequences. Uable 1]

Although the diseases represented in Table 1 rarely occur, it is possible to increase their incidence in the next generation by 50% since some diseases are genetically dormant. Accordingly, in the case of a silent carrier, even though they are not attacked by these diseases, they can transfer the diseases to the next generation without knowledge. In addition, diseases such as Huntington's disease and Myotonic dystrophy develop slowly so that the patient will not realize that he/she has been attacked by the disease until after reproducing the next generation. Accordingly, it is very important to diagnose the multiplication diseases of repeated trinucleotide sequences at an early stage.

Meanwhile, because most neurogenic diseases are caused by expanding and amplifying repeated trinucleotide sequences, said diseases are easily diagnosed or prognosed by monitoring the multiplication level of repeated trinucleotide sequences.

The method used for monitoring the multiplication level of repeated trinucleotide sequences is to measure the length of trinucleotide repeats using molecular genetic techniques. The techniques are exemplified as a Southern blot test and PCR. The Southern blot test is accurate, but it is very expensive and complicated so that it impossible to apply to means for general screening tests.

Meanwhile, PCR amplification techniques are capable of determining the multiplication level of repeated trinucleotide sequences by amplifying trinucleotide repeats. Nevertheless, these techniques have demerits in that it is difficult to accurately determine the size of a PCR amplified product, and it is difficult to find silent carriers.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a simple and accurate method of diagnosing a multiplication disease of repeated trinucleotide sequences.

It is another object to provide a diagnosis kit for simply and accurately analyzing a multiplied level of repeated trinucleotide sequences.

It is further another object to provide a diagnosis device for analyzing a multiplied level of repeated trinucleotide sequences using micro capillary electrophoresis.

It is still another object to provide a nucleotide sequence for specifically amplifying a repeated trinucleotide sequence region.

These and other objects may be achieved by a diagnosis kit for multiplication diseases of repeated trinucleotide sequences, comprising a means for amplifying the repeated trinucleotide sequences using a polymerase chain reaction; and a means for separating the amplified repeated trinucleotide sequence region depending upon the size thereof, wherein the separation means is micro capillary electrophoresis. The amplification means is preferably a primer comprising at least one selected from the group consisting of nucleotide sequences set forth in SEQ. ID. NOs. 1 -26. The present invention further includes a diagnosis device for multiplication diseases of repeated trinucleotide sequences comprising: (a) said diagnosis kit; (b) a means for detecting the migration position of a repeated trinucleotide sequence that is separated by said separation means; and (c) a means for determining the number of repeated trinucleotide sequences on the basis of the results obtained from said detection means.

The present invention further includes a method for diagnosis of multiplication diseases of repeated trinucleotide sequences comprising the steps of: (a) amplifying the repeated trinucleotide sequence region from a DNA sample using a polymerase chain reaction; (b) migrating and separating the amplified product using micro capillary electrophoresis so as to measure the size of the amplified product; and (c) determining the number of repeated trinucleotide sequences on the basis of the size of the amplified product.

The present invention further includes a primer for detecting a repeated trinucleotide sequence comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NOs. 1 -26.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a photograph showing SCA1 , SCA3, SCA6, SCA7, and SCA8 PCR results of healthy subjects; FIG. 1 b is a photograph showing the PCR results of patients with

SBMA;

FIG. 1 c is a photograph showing the PCR results of patients with HD;

FIG. 1 d is a photograph showing the PCR results of patients with Fragile X syndrome;

FIG. 1e is a photograph showing the PCR results of patients with DRPLA, DM, and FRAXE;

FIG. 2 is a photograph showing the PCR results of patient with Fragile X syndrome;

FIG. 3 is a photograph showing the PCR results of patients with SCA3, SCA6, SCA7, and SCA8 PCR results;

FIG. 4 is a photograph showing the PCR results of patients with DM and DRPLA PCR results; FIG. 5 is a photograph showing the SBMA PCR results of healthy subjects and patients;

FIG. 6 is a photograph showing the PCR results of patients with Huntington's disease;

FIG. 7 is a photograph showing the SBMA PCR results and the DOP-PCR results using a DNA sample separated from a pregnant woman's blood;

FIG. 8 is a schematic diagram showing a micro CE chip according to the present invention; and

FIG. 9 is a diagnosis device using the micro CE chip according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors tried to develop a diagnosis method and diagnosis kit for multiplication diseases of repeated trinucleotide sequences, and this was achieved by utilizing micro capillary electrophoresis. The present invention provides a diagnosis method for multiplication diseases of repeated trinucleotide sequences comprising: (a) amplifying the repeated trinucleotide sequence region from a DNA sample using a polymerase chain reaction; (b) migrating and separating the amplified product using micro capillary electrophoresis so as to measure the size of the amplified product; and (c) determining the number of repeated trinucleotide sequences on the basis of the size of the amplified product measured in step (b).

The polymerase chain reaction can be carried out by using a primer specific to the repeated trinucleotide sequence region. The present invention provides primers capable of detecting Huntington's disease (hereinafter referred to as "HD"); Kennedy's disease (hereinafter referred to as "SBMA"); Spinocerebellar ataxia type 1 (hereinafter referred to as "SCAT); SCA2, SCA3, SCA6, SCA7, SCA8, and Dentatorubral- pallidoluysian atrophy (hereinafter referred to as "DRPLA"); Fragile X syndrome (hereinafter referred to as "FRAXA"); Fragile E site (hereinafter referred to as "FRAXE"); Myotonic dystrophy (hereinafter referred to as "DM"); and Friedrich's ataxia (hereinafter referred to as "FA") as shown in the following Table 2. The lengths of PCR amplified products shown in Table 2 correspond to DNA sizes amplified under the absence of repeated trinucleotide sequences when they are subjected to the PCR with a corresponding primer. [Table 2]

Micro capillary electrophoresis (hereinafter referred to as "micro CE") is used to analyze DNA by applying a high voltage to a capillary of 10 jam

-100 μm- That is, DNA is negatively charged so it easily flows toward a

positive electrode upon applying the electro-field. At the same time, DNA will be distributed according to the size and electric charge ratio thereof. Therefore, micro CE is a technique used for separating the DNA using the distribution rate. Micro CE facilitates fabrication of an on-chip analyzing system, since fluid flow is controlled on the micro chip using only the electro-field, so that a fluid control device such as a pump or a valve is not required. In addition, analysis results are obtained in a short time with high separation efficiency. Also, micro CE techniques are being rapidly developed since there are many merits such that the selectivity can be adjusted using various separation modes, it can consume a small amount of sample, it can perform on-capillary detection and quantitative analysis, and it is able to be automated.

Accordingly, the technique using the micro CE chip is advantageous in that the size of PCR product is measured in a short time, and accuracy, reproducibility, and convenience of testing are excellent. In addition, the device will be small and economical since different samples can be analyzed on a chip with a magnitude in the order of several cms.

The representative example of micro capillary electrophoresis may include a micro capillary electrophoresis chip (hereinafter referred to as "micro CE chip"), and preferably a micro CE chip as illustrated in FIG. 8. The micro CE chip may be used in a conventional device used in this field. The micro CE chip shown in FIG. 8 is composed of a channel having a cross shape and a reservoir positioned at the end of the channel to contain a sample and a buffering solution. A sample input reservoir (10) is located at the left end of the channel and a sample output reservoir (12) is located at the right end of the channel. A buffer solution input reservoir (14) is located at the upper end of the channel and a buffer solution output reservoir (16) is located at the lower end of the channel. The channel between the sample input reservoir (10) and the sample out reservoir (12) is an injection channel (22), and the channel between the buffer solution input reservoir (14) and the buffer solution output reservoir (16) is a separation channel (24). The lower part of separation channel (24) is the detection part (26).

In the analysis step, the number of repeated trinucleotide sequences is calculated on the basis of the size of the amplified product measured. Accordingly, prevalence of a multiplication disease of repeated trinucleotide sequences can be diagnosed by comparing the calculated number of repeated trinucleotide sequences with the number of repeated trinucleotide sequences of a healthy subject.

Hereinafter, the process for analyzing the number of repeated trinucleotide sequences is described in more detail. A sample is subjected to the PCR process with a primer having a nucleotide sequence corresponding to each disease. The PCR amplified product is subjected to micro CE to measure the size of the repeated trinucleotide sequence of the sample. Then, the size value is subtracted from the size value of a DNA PCR product of a healthy subject to calculate the number of repeated trinucleotide sequences in the sample.

The present invention further provides a diagnosis kit for multiplication diseases of repeated trinucleotide sequences. The diagnosis kit for the multiplication diseases of repeated trinucleotide sequences comprises a means for amplifying the repeated trinucleotide sequence using a polymerase chain reaction; and a means for migrating and separating the amplified repeated trinucleotide sequence region depending upon the size thereof. The separation means is preferably micro capillary electrophoresis. The amplification means is preferably a primer comprising at least one selected from the group consisting of nucleotide sequences set forth in SEQ ID NOs. 1 -26. Further, the diagnosis kit may further comprise an enzyme and a buffer solution for facilitating a polymerase chain reaction.

The buffer solution for the polymerase chain reaction may include any conventional PCR buffer solution, for example, Tris-HCI, KCI, MgCI₂, or dNTP.

The enzyme for the polymerase chain reaction may include DNA polymerase, and preferably Taq polymerase.

The migration and separation means comprises a micro capillary electrophoresis chip equipped with a channel having a magnitude in the order of μm s and a reservoir on a glass, plastic, or silicone substrate; and a

means for controlling the migration of DNA in the micro capillary electrophoresis chip under the electro-field. It may further comprise a fluorescent dye attached to the repeated trinucleotide sequence separated by the migration and separation means to indicate the migration position of the target.

The sample treated by the diagnosis kit may include a DNA sample separated from whole blood, amniotic fluid, or a nucleated erythrocyte separated from blood obtained from a pregnant woman.

The present invention further provides a diagnosis device for multiplication diseases of repeated trinucleotide sequences comprising the above-mentioned diagnosis kit, a means for detecting a migration position of repeated trinucleotide sequences separated by the migration and separation means; and a means for calculating the number of repeated trinucleotide sequences on the basis of the results obtained from said detection means. The detection means may be an optical or an electrochemical device for detecting a fluorescent dye attached to DNA. The calculation means may comprise a device (e.g., a computer) that is capable of analyzing whether a multiplication disease of a repeated trinucleotide sequence is developed or not by comparing the number of repeated trinucleotide sequences detected by the detection means with that of a healthy subject or of a patient with the multiplication disease of repeated trinucleotide sequences.

One embodiment of the diagnosis kit is shown in FIG. 9. The diagnosis device detects a fluorescent signal of DNA using a laser confocal system. The beam emitted from the laser is transmitted through an excitation filter in order to reduce the noise of the laser itself. The transmitted laser beam enters a dichroic beam splitter which reflects a wavelength region including a laser beam wavelength and passes a wavelength region including a fluorescent wavelength, and focuses onto a target micro channel with an objective lens. When a fluorescent dye or DNA injected with the fluorescent dye migrates to the focus area, the fluorescent dye is excited to emit fluorescence by the light from the laser and is focused by the subjective lens. Since the position for emitting fluorescence is located at the focus of the subjective lens, the fluorescence transmitted through the subjective lens is emitted as a collimated light and is passed through the dichroic beam splitter and focused by the second lens. The focused fluorescence is passed through a pin hole with a diameter in the order of several hundreds of μ - In this case, the light that is not emitted from the focus of the subjective lens (i.e., scattered light of the laser) is not a collimated light so that the light cannot pass through the pin hole. As a result, it can detect only the photo signal emitted from the focus of the subjective lens. The light filtered from the pin hole is passed through an emission filter which passes only a fluorescent wavelength, and it is converted into an electric signal with a Photomultiplier Tube to be recognized by a computer. The laser confocal system receives only the photo signal positioned within the focal length so that it has a very high sensitivity to signal-to-noise ratio. Further, the system will detect very small amounts of fluorescent signal passed though the dichroic beam splitter and the emission filter.

The diagnosis method and the diagnosis kit of multiplication diseases of repeated trinucleotide sequences according to the present invention allow for simple and accurate diagnosis of multiplication diseases of repeated trinucleotide sequences, and they allow diagnosis before the disease developes. Further, it is possible to accurately diagnose whether a silent carrier will develop the disease or not. This method requires only a small amount of sample to diagnose the disease accurately, and it saves costs so that it is possible to apply this method as a general screening test. Further, the present invention may provide a primer for detecting repeated trinucleotide sequences comprising at least one selected from the group consisting of nucleotide sequences set forth in SEQ. ID. NOs. 1 -26.

Hereinafter, the present invention will be explained in detail with reference to Examples. These Examples, however, should not in any sense be interpreted as limiting the scope of the present invention.

Example 1 : Preparation of Primer

A primer including one of the nucleotide sequences set forth in SEQ ID NOs. 1 -26 was individually prepared (Bionic Inc., Canada). Example 2: Screening the Multiplication Disease of Repeated

Trinucleotide Sequences

1 ) Material

DNA of patients with the disease was donated from the Seoul

National University Hospital (Seoul, Korea) and Korean National Institute of Health (Seoul, Korea), and DNA of healthy subjects was obtained from whole blood of healthy humans by using a QiAamp DNA mini kit (QIAGEN

Inc., 28159 Avenue Stanford Valencia CA 91355 USA).

Blood of 16-18 week gravid pregnant women was collected, and fetal

DNA was obtained by separating the single nucleated RBC (snRBC) from

the blood, followed by: dissolving the snRBC in 5 μi distilled water and

immersing it in liquid nitrogen for 5 seconds, three times; adding 5 μi of a

lysis buffer solution (10 mM Tris-HCI, pH 8.3, 50 mM KCI, 500 βg/ i

Proteinase K); and reacting them at 60 ^°C for 2 hours then at 94 ^°C for 2

minutes, to obtain DNA. 2) DOP-PCR

DNA separated from the fetal snRBC was prepared into the following Table 3 PCR compositions, and total genomes are amplified by carrying out the PCR with DOP-PCR primer (SEQ ID NO. 27). [Table 3]

3) Polymerase Chain Reaction (PCR)

The PCR composition of Table 4 was prepared using a primer for screening the repeated trinucleotide sequence, and it was subjected to the

PCR: 95 ^°C , 2 minutes -> (95 ^°C, 30 seconds -> see Table 5, 30 seconds ->

72 ^°C, 30 seconds), 35 times -> 72 ^°C , 3 minutes.

[Table 4]

[Table 5]

The PCR products were analyzed by electrophoresis on 2% agarose gel, and the results are illustrated in FIGs. 1a to 1e. FIG. 1a is an electrophoresis photograph of DNA of healthy subjects that were subjected to the PCR process with a primer set for screening SCA1 , a primer set for screening SCA3, a primer set for screening SCA6, a primer set for screening SCA7, and a primer set for screening SCA8. Lane 1 is represented as a 100 bp ladder, lanes 2 and 3 are PCR products amplified by a primer set for screening SCA1 , lanes 5 and 6 are PCR products amplified by a primer set for screening SCA3, lanes 8 and 9 are PCR products amplified by a primer set for screening SCA6, lanes 11 and 12 are PCR products amplified by a primer set for screening SCA7, and lanes 14 to 16 are PCR products amplified by a primer set for screening SCA8. Lanes 4, 7, 10, 13, and 16 are negative controls. FIG 1b is an electrophoresis photograph of DNA of patients with

SBMA who were subjected to the PCR with a primer set for screening SBMA.

Lane 1 is represented as a 100 bp ladder, lanes 2 to 14 are PCR products using templates of DNA of SBMA patients, and lane 15 is a negative control.

FIG 1c is an electrophoresis photograph of DNA of patients with HD who were subjected to the PCR with a primer set for screening HD. Lane 1 is represented as a 100 bp ladder, lanes 2 to 10 are PCR products using templates of DNA of HD patients, and lane 11 is a negative control.

FIG 1d is an electrophoresis photograph of DNA of patients with

FRAXA who were subjected to the PCR with a primer set for screening FRAXA. Lane 1 is represented as a 100 bp ladder, lane 2 is a negative control, and lanes 3 to 21 are PCR products using templates of DNA of healthy subjects.

FIG 1e is an electrophoresis photograph of DNA of patients with DRPLA, DM, or ERAXE who were subjected to the PCR. Lane 1 is represented as a 100 bp ladder, lanes 2 to 5 are PCR products using templates of DNA of DRPLA patients, lanes 7 to 11 are PCR products using templates of DNA of DM patients, lanes 13 and 14 are PCR products using templates of DNA of FRAXE patients, and lanes 6, 12, and 15 are negative controls. 4) Analysis of DNA nucleotide sequence of amplified product

The nucleotide sequences of the PCR amplified products obtained from the above examples were analyzed with an automatic sequencer.

Each PCR amplified product was purified with a PCR purification kit (QIAquick PCR purification kit, QIAGEN, USA), and subjected to sequencing (with an ABI PRISM™ Dye Terminator Cycle Sequencing kit). A PCR nucleotide sequencing reaction was performed using a GeneAmp PCR system 9600, model 9600 Perkin-Elmer (Foster city, CA, USA) with 25 cycles at 96 ^°C for 30 seconds, at 50 °C for 15 seconds, and at 60 °C for 4 minutes.

Unreacted nucleotides were removed using Centri-Sep™ spin columns (Princeton Separations, Philadelphia, NJ, USA) and dried using Speed Vac

(Savane Instruments, Farmingdate, NY, USA), followed by dissolving in 6 μi

of a loading buffer (deionized formamide 5 μi, 25mM EDTA (pH 8.0)

containing 50 mg/mβ blue dextran 1 μi). It was denatured immediately

before loading at 90 ^°C for 2 minutes, and each 15 μi was subjected to

electrophoresis with 15 KW on 4.75% denatured polyacrylamide gel. The nucleotide sequence was then analyzed using an ABI PRISM™ 310 Genetic analyzer.

5) Analysis using Micro CE chip PCR results were respectively analyzed on 2% agarose gel and the micro CE chip.

A DNA 500 LabChip capable of detecting in a range of DNA 25-500

bp was used, and the temperature was constantly maintained at 30 ^°C . Gel

(Agilent) and dye were mixed and loaded into the chip, and 1 μi of a DNA

500 ladder was inserted into a ladder hole. The dye was attached to the DNA so that the DNA was detected when it migrated on the micro CE chip.

Hereinafter, 1 μi DNA and 5 μi of a size marker (15/600 bp) were inserted

into a separation hole. The chip was stirred in the vortex and introduced into an Agilent 2100 Bioanalyzer (USA) and subjected to electrophoresis. FIG. 2 illustrates PCR results of a healthy subject using a primer for screening Fragile X syndrome, and Table 6 shows the size of PCR products analyzed using a sequencer and the number of repeats of the trinucleotide sequence. [Table 6]

FIG. 3 illustrates PCR results using a primer for screening SCA3, SCA6, SCA7, and SCA8, and Table 7 shows the size of PCR products analyzed using a sequencer and the number of repeats of the trinucleotide sequence. [Table 7]

FIG. 4 illustrates PCR results using a primer for screening DM or a primer for screening DRPLA, and Table 8 shows the size of PCR products analyzed using a sequencer and the number of repeats of the trinucleotide sequence.

[Table 8]

FIG. 5 illustrates PCR results using a primer for screening SBMA, and Table 9 shows the size of PCR products analyzed using a sequencer and the number of repeats of the trinucleotide sequence. [Table 9]

FIG. 6 illustrates PCR results using a primer for screening HD, and

Table 10 shows the size of PCR products analyzed using a sequencer and the number of repeats of the trinucleotide sequence. [Table 10]

FIG. 7 illustrates PCR results of DNA separated from pregnant women's blood using a primer for screening SBMA and DOP. In FIG. 7, lane 1 is represented as a 100 bp marker, lane 2 illustrates the SNMA PCR result, and lane 3 illustrates the DOP-PCR result. The DOP-PCR amplification product amplified from the whole genome was revealed as a smear on the gel. The size of the lane 2 product was measured as 272 bp, which corresponds to 17 repeats of the trinucleotide sequence.

Example 3: Preparation of Diagnosis Device using a Micro CE Chip

The micro CE chip as shown in FIG 8 was prepared as follows. A channel with a width of 70 μm and a depth of 15 μm, and a

separating channel with a length of 38 mm were prepared on a glass wafer (0.5 mm thick Schott Borofloat). The wafer was covered with a Cr/Au mask and was spin-coated with a positive photoresist (AZ 1512) at 500 rpm for 5 seconds and at 4000 rpm for 30 seconds. The wafer coated with the photoresist (PR) was soft baked at 90 ^°C for 30 minutes, followed by exposure to UV light through the Cr photo mask to make a desired pattern on the wafer. The exposing duration was 10 seconds. After developing

the photoresist, it was hard-baked at 1 15^°C for 30 minutes. Then, Au was

removed with aqua regia within several seconds, and Cr was removed with a Cr7 etching solution. The channel was etched with a buffer HF solution. A cover glass (0.5 mm thick, Schott Borofloat) having a cavity with a diagram of 15 mm located at the position corresponding to the each reservoir was prepared, then the cover glass was thermal connected with the wafer having a channel. Acrylamid was added to 45 mM tris/45 mM borate/1 mM EDTA/8.3 M urea (pH 8.3) and injected into the channel, then the polymerization was

carried out by adding 2.5 μi of 10% ammonium persulfate and TEMED.

To perform the electric analysis and the detection, a MP5P24 power supply available from Spellman (US) and a laser detector that was set up by the inventor were used. To control the voltage, a LabVIEW (National Instruments, USA) may be used. FIG. 9 shows a schematic diagram of a diagnosis device for multiplication diseases of repeated trinucleotide sequences according to the present invention.

Hereinafter, the DNA separation mechanism is described in brief. The sample reservoir was applied with a high voltage prior to loading the sample. The sample output reservoir was grounded to make the sample migrate. To prevent inducing the sample into the separation channel, the buffer input solution reservoir and buffer solution output reservoir were applied with a high voltage so that the sample was controlled to migrate only toward the sample output reservoir. On loading the sample, an electric field of 170 V/cm was supplied to the sample input channel for 60

seconds then cut off immediately, and the separation was carried out by applying an electric field of 250 V/cm to the separation channel. In other

words, the sample in the sample input reservoir was flowed toward the separation channel in accordance with the flowing of the buffer solution by changing the voltage gradient, and thereby unfavorable diffusion of the sample was minimized so as to achieve a substantially accurate sample loading process. As mentioned above, if the high voltage is supplied to the channel, DNA having negative charge characteristics migrates toward the positive electrode. During the migration, DNA is separated at a rate distribution that is generated according to the size of the DNA and the charge ratio.

Claims

WHAT IS CLAIMED IS:

1 . A diagnosis kit for a multiplication disease of a repeated trinucleotide sequence, comprising a means for amplifying the repeated trinucleotide sequence using a polymerase chain reaction; and a means for migrating and separating the amplified repeated trinucleotide region depending upon the size thereof, wherein the separation means is micro capillary electrophoresis.

2. The diagnosis kit according to claim 1 , wherein the amplification means is a primer comprising at least one selected from the group consisting of nucleotide sequences set forth in SEQ ID NOs. 1 -26.

3. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 1 or 2 is used to detect Huntington's disease.

4. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 3 or 4 is used to detect Kennedy's disease.

5. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 5 or 6 is used to detect Spinocerebellar ataxia 1 .

6. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 7 or 8 is used to detect Spinocerebellar taxia 2.

7. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 9 or 10 is used to detect Spinocerebellar ataxia 3.

8. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 11 or 12 is used to detect Spinocerebellar ataxia 6.

9. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 13 or 14 is used to detect Spinocerebellar ataxia 7.

10. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 15 or 16 is used to detect Dentatorubral pallidoluysian atrophy.

11. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 17 or 18 is used to detect Fragile X syndrome.

12. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 19 or 20 is used to detect Fragile X site.

13. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 21 or 22 is used to detect Myotonic dystrophy.

14. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 23 or 24 is used to detect Friedrich's ataxia.

15. The diagnosis kit according to claim 2, wherein the primer comprising a nucleotide sequence set forth in SEQ ID NO. 25 or 26 is used to detect Spinocerebellar ataxia 8.

16. The diagnosis kit according to claim 1 , wherein the DNA sample is separated from whole blood, amniotic fluid, or nucleated erythrocyte separated from blood obtained from a pregnant woman.

17. The diagnosis kit according to claim 1 , further comprising an enzyme and a buffering solution for polymerase chain reaction.

18. The diagnosis kit according to claim 1 , wherein the migration and separation means comprise: a micro capillary electrophoresis chip equipped with a channel

having a magnitude in the order of m s and a reservoir on a glass, plastic,

or silicone substrate; and a means for controlling the movement of DNA in the micro capillary electrophoresis chip under an electro-field.

19. The diagnosis kit according to claim 18, further comprises fluorescein attaching to the repeated trinucleotide sequence separated by the migration and separation means to indicate the moved position thereof.

20. A diagnosis device for a multiplication disease of a repeated trinucleotide sequence, comprising:

(a) the diagnosis kit according to any one of claims 1 to 19; (b) a means for detecting a moved position of the repeated trinucleotide sequence separated by the migration and separation means; and

(c) a means for calculating the number of repeated trinucleotide sequences on the basis of the results obtained from said detection means.

21. The diagnosis device according to claim 20, wherein the detection means is an optical or an electrochemical device for detecting fluorescein attached to DNA.

22. The diagnosis device according to claim 20, wherein the calculation means comprises a computer for calculating whether the multiplication disease of trinucleotide repeated sequences is developed or not by comparing the number of repeated trinucleotide sequences detected by the detection means with that of a patient with the multiplication disease of repeated trinucleotide sequences or with that of a healthy subject.

23. A diagnosis method of a multiplication disease of repeated trinucleotide sequences comprising the steps of:

(a) amplifying the repeated trinucleotide sequence region from a DNA sample using a polymerase chain reaction;

(b) migrating and separating the amplified product using micro capillary electrophoresis so as to measure the size of the amplified product; and

(c) calculating the number of repeated trinucleotide sequences on the basis of the size of the amplified product.

24. The diagnosis method according to claim 23, wherein the amplification step is carried out by amplifying the repeated trinucleotide sequence region with a primer comprising at least one selected from the group consisting of nucleotide sequences set forth in SEQ ID NOs. 1 -26.

25. A primer for detecting a repeated trinucleotide sequence comprising at least one selected from the group consisting of nucleotide sequences set forth in SEQ ID NOs. 1 -26.