KR20020067256A - Microarray system using chamber slide that has spotted bio-polymer separately - Google Patents

Abstract

PURPOSE: Provided is a microarray system using chamber slide having spotted bio-polymer separately to diagnose various specimens obtained from several persons with only one microarray slide. CONSTITUTION: The microarray system is characterized by containing the steps of: manufacturing a biochip by spotting biopolymers which are partitioned on a chamber slide; equipping walls on the chamber slide then loading samples thereinto; and removing the walls then assaying reactivity.

Description

MICROARRAY SYSTEM USING CHAMBER SLIDE THAT HAS SPOTTED BIO-POLYMER SEPARATELY}

The present invention relates to a microarray system for compartmentalizing and integrating a biopolymer into a chamber slide, and more particularly, to a microarray system capable of compartmentalizing and integrating cDNA, oligonucleotide or protein.

Many genes are amplified by PCR and then integrated on glass slides using robotic arrays, called microarrays, also known as DNA chips.

Hybridization-based microarray analysis is one of the most widely used techniques in recent years, and has many applications. It is gradually being developed from the basic concept of detecting nucleic acid molecules immobilized on. Numerous venture companies are now involved in the production and application of DNA chips, with giants including Molecular Dynamics and Motorola. Until 1998, the only array available to the general researcher was a form in which genes were fixed on a filter. However, NEN Life Science launched a product in which 2,400 human cDNAs were fixed on slide glass, which is a representative type of DNA chip. , Incyte et al. Commercialized DNA chips for human EST, mouse, yeast and several bacteria, and Clontech has also released slide-type cDNA arrays.

These microarrays can be tested for a variety of genetic information can be easily analyzed from disease diagnosis of the patient to genetic factor information of the animal. However, currently used microarrays use non-partitioned glass slides, so that only one sample can be analyzed by accumulating the same kind of biomaterial on one slide, which limits the use of expensive microarrays. It is true. If a slide can be partitioned, it is possible to use a microarray much more economically by dividing a slide into sections and testing various samples, that is, several samples, with the same type of biomaterial.

Accordingly, an object of the present invention is to provide a microarray system capable of partitioning and integrating cDNA, oligonucleotide or protein.

It is another object of the present invention to provide a slide for a microarray capable of integrating various kinds of cDNA, oligonucleotide or protein set in one slide.

1 shows a chamber slide of the present invention,

2 is a cross-sectional view of a biochip using the chamber slide of the present invention.

The present invention to achieve the above object

(a) manufacturing a biochip by dividing and spotting the biopolymer with the chamber of the chamber slide removed;

(b) installing a storage chamber in the chamber slide, and putting a sample into each compartment for reaction; And

(c) removing the chamber of the chamber slide after the reaction and then checking the reactivity of the microarray;

It provides a microarray system comprising a.

The present invention also provides a biochip in which a biopolymer is partitioned and integrated on a microarray slide.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for compartmentalizing and integrating biopolymers in a biochip or microarray system. The biopolymer refers to any kind of material that can be integrated in a biochip, and is preferably cDNA, RNA, oligonucleotide, protein or chemical.

The microarray system for partitioning and integrating the biopolymer of the present invention is achieved by using a chamber slide. One example of a chamber slide of the present invention is shown in FIG. The chamber slide is characterized in that it comprises a storage chamber (2) comprising at least three sides formed with a space for containing the contents on the surface of a conventional microarray slide (1). In addition, the storage chamber is installed to be detachable. It is preferable to adjust the number of accommodation chambers suitably according to application use, and it is more preferable to use four, eight, or 16 accommodation chambers. Since the chambers of the chamber slide are provided with a silicon wire 3 connecting the chambers, it is preferable that the chambers be operated integrally when the chamber is attached or detached.

The microarray system of the present invention comprises the steps of (a) manufacturing a biochip by dividing and spotting a biopolymer in a state in which a chamber of a chamber slide is removed, (b) installing a chamber in the chamber slide, Putting the sample into the compartment for reacting, and (c) removing the chamber of the chamber slide after the reaction and then checking the reactivity of the microarray.

In the step (a), the biopolymer (cDNA, oligonucleotide, protein, or chemical) may be accumulated in a plurality of storage chambers. The method of integrating is preferably carried out in a conventional manner according to the biopolymer material.

During the sample reaction of step (b), the reaction between the sample and the biopolymer is carried out on the storage chamber, so that the reaction space is sufficient, the accuracy is high, and the different samples can be reacted between the storage chambers, thereby allowing a plurality of samples to be accurately reacted. In addition, multiple diseases can be diagnosed with one sample. The microarray slides used at this time should be integrated with biopolymers that can diagnose different kinds of diseases in each chamber.

After the step (b), the washing process or the detection process may be further performed with the storage chamber removed, and the microarray reactivity of step (c) may be performed using conventional equipment.

The present invention also provides a biochip using a chamber slide. That is, the chamber slide may be provided as a separate biochip without mixing between biomaterials by spotting various types of biomaterials (DNA, RNA, oligonucleotides, proteins or chemicals) in each storage chamber.

As mentioned above, the microarray system of the present invention can check the reaction degree of a biochip by using a conventional biochip scanner by removing a storage chamber by reacting a sample in each chamber and then removing the chamber. The process except the reaction with the heavy sample can be carried out with the storage chamber removed, so that the existing biochip operating system can be used as it is. In addition, different samples can be simultaneously reacted on one biochip.

The present invention also provides a biochip in which a biopolymer is partitioned and integrated on a chamber slide. The biochip has an advantage that can react a plurality of samples at one time. One example of a bio pip is shown in FIG. 2.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1

On the slide surface of 7.5 cm x 2.5 cm glass, four rectangular storage chambers consisting of four sides of 2 cm x 1 cm x 1 cm (width x length x height) were detachably installed. The accommodating chamber is provided with a silicon wire to connect the accommodating chamber. In addition, a chamber cover was prepared at 4.6 cm x 2.2 cm x 0.5 cm to cover the chamber slide.

Example 2 cDNA Oligonucleotide Microarray Preparation

The lattice of the chamber slide was removed, the poly-L-lysine was coated, the DNA dissolved in the secondary distilled water was spotted on the chamber slide, and the lattice of the slide was installed as it was.

Experimental Example 1

sample preparation

MRNA was extracted from the assay sample in a conventional manner and cDNA was synthesized using reverse transcriptase. CDNA was labeled with Cy3-dTP or Cy5-dTTP during the synthesis. 0.1 N NaOH was added to the synthesized cDNA and reacted at 70 ° C. for 10 minutes, followed by neutralization by mixing 0.1 N HCl. 20 ug of cot 1 human DNA was added thereto, and the probe was purified by Centricon-30 microcentrifuge. Add a labeled probe to the new centricon and dilute to 0.1 ug / ul.

detection

200 ul each of the probes prepared in the DNA chip prepared in Example 2 and sealed the upper surface of the chamber slide using 3M tape. The chamber slide was reacted at 65 ° C. for 14 to 18 hours, the probe was removed, and then the lattice was removed. Wash once, twice, and three times with 1st wash solution (1X SSC / 0.03% SDS), 2nd wash solution (0.2X SSC) and 3rd wash solution (0.05X SSC), respectively. The washed slides were spun-dryed by centrifugation at 650 rpm for 2 minutes and placed in a scanner to confirm the detection result.

Example 3 Preparation of Protein Microarray

Protein was dissolved at 100-200 ug / ml in PBS / 0.02% sodium azide buffer. The chamber slides removed the lattice and the slides were treated with poly-L-lysine. Protein lysates were accumulated on the slides and soaked several times in washing solution (PBS, 3% nonfat milk, 0.1% Tween 20). Immediately thereafter the slides were transferred to blocking solution (PBS, 3% nonfat milk, 0.2% sodium azide) and reacted overnight at 4 ° C. Thereafter, the slides were washed three times in succession for one minute with 0.2 X PBS solution in order to remove the milk that did not adhere on the slides, and soaked in washing buffer (0.2 X PBS) until use. The grid was installed on the slide before use.

Experimental Example 2

Sample proteins to be measured were prepared on 0.1 M carbonite or phosphate buffer (pH 8.0) solution at a concentration of 15 ug / 200 ul per array. Cy-stained with NHS-ester (Amersham buffer, pH8.0) was added to the protein solution at a concentration of 100-300 uM and mixed. The control group was reacted with Cy3 stain, and the sample protein was reacted with Cy5 stain for 45 minutes at room temperature. The reaction was stopped by mixing 1 M Tris or glycine with the reaction, and the reaction product was obtained by removing dye using a molecular weight blocking microcon (3000D). The protein was centrifuged (10,000xg, 80) according to the microcon manual. Min, room temperature). 3% blocking solution was added to the separated protein solution, and 500 ul of PBS was added, followed by centrifugation. 5 ul of concentrated sample protein was obtained, and then a control protein solution labeled with Cy3 and a sample protein solution labeled with Cy5 were mixed and PBS was added.

detection

The array was removed from the PBS solution and then the solution was removed to treat the labeled protein mixture on the array. The chamber slides were sealed with 3M tape on a lattice and placed in a wet chamber for 2 hours at 4 ° C. The chamber slide was removed from the chamber to remove the lattice and then placed in a slide rack containing PBS / 0.1% Tween 20 solution and stirred for 20 minutes on a rotary stirrer. The chamber slides were transferred to a new rack and then washed with PBS for 5-10 minutes and twice with water for 5 minutes.

As mentioned above, the microarray system of the present invention can partition the material to be integrated on a slide and can simply use various kinds of materials by integrating on one slide. It can also be confirmed by applying different kinds of samples with one microarray.

Claims (8)

(a) manufacturing a biochip by dividing and spotting the biopolymer with the chamber of the chamber slide removed; (b) installing a chamber in the chamber slide, and putting a sample into each chamber for reaction; And (c) removing the chamber of the chamber slide after the reaction and then checking the reactivity of the microarray; Microarray system comprising a. The microarray system according to claim 1, wherein the chamber slide is provided with a storage chamber in which a space for containing contents is formed on a surface of a microarray system slide. The microarray system according to claim 2, wherein the chambers of the chamber slides are installed so that materials in each chamber are independently present, and the chambers are detachably mounted on the microarray slides. The microarray system of claim 1, wherein the biopolymer is spotted with at least one selected from the group consisting of cDNA, protein, oligonucleotide and chemicals. The microarray system according to claim 1, wherein step (a) divides the biopolymer into a plurality of compartments, and step (b) reacts different types of samples in each compartment. The method of claim 1, wherein step (a) comprises partitioning and accumulating different types of biopolymers that can identify a plurality of diseases, and step (b) reacts a sample in each compartment to make a plurality of diseases in one microarray slide. Microarray system, characterized in that for diagnosing. A biochip in which biopolymers are partitioned and integrated on a microarray slide. The biochip according to claim 7, wherein the biochip has at least one spot selected from the group consisting of DNA, RNA, protein, polynucleotide, and chemicals.