KR20150049256A - Probes, DNA chip and kit for HLA typing specific to rheumatism, and method for genotyping HLA alleles specific to rheumatism using the same - Google Patents

Abstract

The present invention provides a method for simply, rapidly, and accurately analyzing an HLA-DRB1 allelotype while a subtype represented by a four-digit number of a rheumatism-related HLA-DRB1 allelotype is discriminated in a high resolution. According to the present invention, time taken to analyze a sample is remarkably reduced compared to an existing method, and a large quantity of samples can be inspected in a short time, so that HLA-DRB1 allelotypes can be mass-analyzed in a high resolution. The occurrence of rheumatism-related diseases and dangers can be accurately and effectively diagnosed and predicted in a genetic level. Moreover, in the present invention an HLA-DRB1 allelotype analysis in a Korean group can be performed, and an HLA-DRB1 allelotype pattern related to rheumatism-related diseases of Koreans can be determined. Therefore, the same has a high utilization value in a health and medical manner for treating and preventing rheumatism diseases.

Description

TECHNICAL FIELD The present invention relates to a probe composition for HLA typing specific to rheumatism, a DNA chip and kit, and a method for analyzing rheumatism-specific HLA-DRB1 allele genotypes using the same. rheumatism using the same}

The present invention relates to a probe composition for HLA typing specific to rheumatism, a DNA chip and kit, and a method for analyzing rheumatism-specific HLA-DRB1 alleles using the same, and more particularly, A DNA chip and kit, and a rheumatism-specific HLA-DRB1 allele genotyping method using the probe composition, the DNA chip and the kit, for the rheumatism-specific HLA typing.

Specifically, the present invention relates to a probe composition for HLA typing specific to rheumatism, which can analyze HLA-DRB1 allele genotype related to rheumatism in high resolution and can perform HLA-DRB1 allele genotype analysis simply, quickly, DNA chip and kit, and a method for analyzing rheumatism-specific HLA-DRB1 allelic genotypes using the same.

Rheumatism is an acute or chronic disease that manifests pain, movement disorders, and induration in muscles, joints, or adjacent tissues. Rheumatologic diseases, including joint rheumatism and neuralgia, are exacerbated by movement disorders and often lead to post-eventual cardiac valve disease. During such repeated and repeated rheumatic symptoms, the illness is worsened, resulting in movement disorders and cardiac disorders.

There are many kinds of diseases belonging to rheumatism and many patients, especially chronic rheumatoid arthritis. Rheumatoid arthritis appears in the serum of patients with rheumatism. After the fact that it is known that there is a self-antibody against IgG (immunoglobulin G), it is thought to be caused by immune disorders.

The prevalence of rheumatoid arthritis, a typical disease of rheumatoid disease, is generally known to be about 1% of the total population, and it is known that the prevalence of women is 2-4 times higher than that of men and increases with age.

Rheumatoid arthritis is a typical systemic chronic autoimmune disease involving joints, which is difficult to cure and causes joint pain, deformation and disability as well as indirect costs due to loss of labor force, which is a very socio-economic cost burden.

However, rheumatoid arthritis is predictive of diagnosis and disease development through various autoantibody tests such as anti - CCP factors or rheumatoid factor. However, there is a limit to make a judgment based on clinical features and laboratory findings. In particular, it is difficult to predict whether or not a disease occurs in an autoantibody-positive group without symptoms. In the case of arthritis without an autoantibody, diagnosis is delayed and there is a problem in missed treatment period. .

Recently, a combination of autologous antibodies (RF and ACPA), CRP (C reactive protein), and joint damage score (Erosion score), like the matrix risk model, predicts the prognosis of rheumatoid arthritis, Although efforts are being actively made to present standards, there is ambiguity with other immune diseases, and there is still a limit to using only a small number of clinical information.

Recent studies have shown that HLA typing, which has been identified as a major test item in tissue and bone marrow transplant surgery, has been shown to have diagnostic potential in autoimmune diseases such as rheumatoid arthritis and is based on serologic methods for HLA antigens Inspection methods have been developed.

The characteristics of HLA (human leukocyte antigen) include polymorphism, which is the individual difference in the immune response to a specific peptide, which determines the individual difference in susceptibility to autoimmune diseases, HLA typing that detects it is very important because it induces the reaction. The HLA allele is a structure that is determined only at the DNA level and is given a permanent name, followed by an asterisk followed by a four-digit number, with the first two digits of the number corresponding to the previous serological typing, Which is subdivided into subtypes. For example, DRB1 * 0405 indicates the fifth subtype of the allele corresponding to the previous DR4 allele.

As molecular biology advances, the nucleotide sequence of the allele that controls the HLA antigen has been elucidated. As a result, HLA gene analysis that verifies the diversity of the allele genotype of HLA has become possible by the difference of the base sequence. A methodological approach to diagnosis and predictability of genetic level of autoimmune diseases such as rheumatoid arthritis has been suggested.

 The introduction of the PCR method has led to radical development of the DNA typing field of HLA, and almost all the polymorphisms of the HLA antigen have been revealed at the DNA level. However, the conventional PCR method used for analyzing the HLA gene, pyrosequencing, Have required a great deal of time and labor to simultaneously type HLA-A, -B, -Cw, and -DRB1, and are inadequate for analyzing a large number of specimens. Therefore, in recent years, a DNA chip technology capable of attaching a very small amount of DNA onto a substrate of a microscopic size such as a slide glass or a silicon wafer at a high density and simultaneously detecting a large number of DNA chips can be used for gene expression analysis, gene diagnosis, And so on. However, such DNA chip technology is also used for HLA typing, which can overcome the disadvantages of the prior art as described above.

In this regard, in Korean Patent Registration No. 10-0581002, HLA-A, -B, -Cw, and -DRB1 are synthesized using the principle of a DNA chip that can overcome the disadvantages of the conventional HLA typing method described above. Discloses a low resolution DNA chip that analyzes simultaneously on one slide. Korean Patent Registration No. 10-0581002 discloses a method for synthesizing HLA-A, -B, -Cw, and -DRB1 corresponding probes on one aldehyde glass slide and amplifying the HLA gene amplified using the asymmetric PCR method The hybridization reaction was performed, and the results of the probe in which the reaction was indicated were analyzed for HLA allele genotypes using a program. However, even though the HLA typing DNA chip of Korean Patent Registration No. 10-0581002 is a superior technology, it can be continuously improved like other superior technologies. Namely, Korean Patent Registration No. 10-0581002 discloses only a low-resolution DNA chip capable of discriminating the HLA allele genotype only in two-digit units, and can not discriminate the HLA allele genotype having various subtypes at high resolution, And prediction.

Therefore, in the technical field to which the present invention belongs, the HLA-DRB1 allele genotype related to rheumatism is discriminated at a high resolution up to the subtype represented by 4 digits, and the HLA-DRB1 allele genotype analysis is performed simply, quickly, There is a continuing need for the development of technologies that can accurately and effectively diagnose and predict disease status and risk at the genetic level.

Korean Registered Patent No. 10-0581002 (2006. 05. 17)

The present invention relates to a probe composition, a DNA chip and a kit for HLA typing specific to rheumatism capable of discriminating rheumatoid related HLA-DRB1 allele at high resolution up to the subtype represented by 4-digit number, and a rheumatic specific HLA-DRB1 It is an object of the present invention to provide an allelic genotyping method.

Specifically, the present invention relates to a probe composition, DNA chip and kit for HLA typing specific to rheumatism, which can perform simple, rapid and accurate analysis of HLA-DRB1 allele genotype while analyzing allele genotype of rheumatoid related HLA-DRB1 allele genotype And to provide a method for analyzing rheumatism-specific HLA-DRB1 allelic genotypes using the same.

In addition, the present invention can greatly reduce the time for analyzing a sample compared to the conventional method, and can test a large amount of samples in a short time, and it is possible to carry out a large-scale HLA-DRB1 allele genotype analysis with high resolution, And to provide a technique for accurately and effectively diagnosing and predicting the onset and risk of a disease at a genetic level.

In addition, the present invention is capable of performing HLA-DRB1 allelic genotyping in a Korean population, thereby determining the HLA-DRB1 allele pattern associated with rheumatic diseases in Koreans, The goal is to provide technology that is highly utilized in the dimension.

The present invention provides a simple, rapid, and accurate method for performing HLA-DRB1 allele genotype analysis while distinguishing rheumatoid related HLA-DRB1 alleles at a high resolution up to a four-digit subtype level. The present inventors analyzed the HLA-DRB1 allele genotype and the single nucleotide polymorphism (SNP) associated with rheumatoid development prediction found in Koreans through a variety of researches and efforts, and selected contents for a DNA chip for high resolution HLA typing, As a result of intensive research based on this research, eight HLA-DRB1 alleles (* 0101, * 0401, * 0404, * 0405, * 0408, * 0410, * 0901, * 1001) related to rheumatism were selected.

Because each of the eight HLA-DRB1 alleles associated with rheumatism (* 0101, * 0401, * 0404, * 0405, * 0408, * 0410, * 0901, * 1001) There is a problem that the specificity and the sensitivity can not be satisfied at the same time when the probe is designed. That is, when individual probes are prepared for each of the eight HLA-DRB1 alleles to obtain a probe detection sensitivity higher than a predetermined cutoff value, each of these probes is subjected to the HLA-DRB1 antagonism due to the individual diversity of the HLA allele In addition to the gene, it also binds to other HLA-DRB1 alleles, generating a false-positive signal.

Therefore, in the present invention, a combination set of optimal probes suitable for typing the eight HLA-DRB1 alleles (* 0101, * 0401, * 0404, * 0405, * 0408, * 0410, * 0901, * 1001) And analyzed the allele genotype of HLA-DRB1 according to the signal pattern from these probe combinations.

In view of such circumstances, the probe composition for HLA typing specific to rheumatism of the present invention of the present invention comprises an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide And at least one probe selected from the group consisting of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having the nucleotide sequence complementary to the oligonucleotide, at least one probe selected from the group consisting of the nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide having a sequence and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5, At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6 and an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6, and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide, and at least one probe having a nucleotide sequence of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide, at least one probe selected from the group consisting of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 9, An oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 10, and an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 10, and at least one probe selected from the group consisting of an oligonucleotide having a base sequence complementary to the oligonucleotide Gt; probe.

The DNA chip for HLA typing specific to rheumatism in one embodiment of the present invention comprises at least one selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide And at least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4, At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence complementary to an oligonucleotide, an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5, and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide And at least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 6 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide, and at least one probe selected from the group consisting of SEQ ID NO: At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide, an oligonucleotide having a nucleotide sequence of SEQ ID NO: 8, and an oligonucleotide complementary to such oligonucleotide An oligonucleotide having a nucleotide sequence of SEQ ID NO: 9, and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide, wherein the oligonucleotide has a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: And at least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 10 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide, and at least one probe selected from the group consisting of .

In a DNA chip for HLA typing specific to rheumatism in an embodiment of the present invention, the probe may be microarrayed on the same substrate such as a glass slide. It is preferable that the substrate is a glass substrate or a plastic substrate generally used for producing DNA chips. It is also preferable that the DNA chip in which the probe is microarrayed is additionally fixed with a position marker indicated by a specific nucleotide sequence so as to enhance the accuracy of hybridization and detection with the probe.

The PCR amplification kit specifically amplifying the rheumatoid-related HLA-DRB1 allele of one embodiment of the present invention comprises the forward primers of SEQ ID NOs: 11 to 14, the primer set of the reverse primer of SEQ ID NO: 15, the DNA polymerase, dNTPs, PCR buffer solutions, and labeled substances of PCR amplification products.

A method for analyzing rheumatism-specific HLA-DRB1 allelic genotypes according to an embodiment of the present invention comprises the steps of amplifying a sample DNA by performing PCR and amplifying the PCR amplification product on a DNA chip for HLA typing specific to rheumatism , And analyzing the HLA-DRB1 allele according to the hybridization status.

The rheumatism-specific HLA-DRB1 allele genotyping method of the embodiment of the present invention can discriminate the rheumatoid related HLA-DRB1 allele to the four-digit subtype.

In the method for analyzing alleles of HLA-DRB1 alleles according to an embodiment of the present invention, the step of analyzing the HLA-DRB1 allelic genotype includes hybridizing fluorescence-labeled PCR amplification products to the probes, Assigning a value of "1" for a probe representing a cutoff value or more and assigning a value of "0" for a probe for which a fluorescence intensity is less than a cutoff value, Generating a binary code composed of "1" and "0 " with respect to the HLA-DRB1 gene; and matching each generated binary code with an allele of HLA-DRB1 to be analyzed.

In a method for analyzing rheumatism-specific HLA-DRB1 allelic genotypes of an embodiment of the present invention, a combination of binary codes of allele 1 and allele 2 present on diploids is generated, and a combination of these binary codes is used to generate HLA- Characterized by analyzing whether the DRB1 allelic genotype is homozygote or heterozygote.

In addition, the present invention provides a test kit capable of analyzing rheumatism-specific HLA-DRB1 allele.

Specifically, the test kit capable of analyzing the rheumatism-specific HLA-DRB1 allele genotype of the present invention comprises a DNA chip defined as described above, and a DNA chip defined as SEQ ID NO: 11 for specifically amplifying a rheumatism-related HLA-DRB1 allele A primer set consisting of forward primers of SEQ ID NO: 14 and reverse primer of SEQ ID NO: 15, and labeling means for detecting amplified gene products hybridized with the DNA chip.

Meanwhile, in the present invention, the labeling substance is not limited to a specific fluorescent substance, and various fluorescent substances which can be confirmed as a fluorescence detector can be used, and an enzymatic coloring reaction and isotope labeling are also possible. For example, the labeling substance may be Cy5, Cy3, a biotin-binding substance, EDANS (5- (2'-aminoethyl) amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate ), x-rhodamine, or Texas red, and is preferably Cy3 or Cy5 which is a fluorescent material. However, the present invention is not limited thereto, and various labeling materials such as various fluorescent materials, radioactive materials, and luminescent materials known in the art can be used.

According to the present invention, it is possible to discriminate the rheumatoid-related HLA-DRB1 allele at high resolution up to the four-digit subtype level, and to analyze the rheumatoid related HLA-DRB1 allele with high resolution, Analysis can be performed.

In addition, according to the present invention, a large amount of samples can be inspected in a short time while greatly shortening the time for analyzing a sample as compared with the conventional method, and it is possible to perform a large-scale HLA-DRB1 allele genotype analysis with high resolution, It has the advantage of accurately and effectively diagnosing and predicting the onset and risk of the disease at the genetic level.

In addition, the present invention is capable of performing HLA-DRB1 allelic genotyping in a Korean population, thereby determining the HLA-DRB1 allele pattern associated with rheumatic diseases in Koreans, It can be said that the value of utilization is high.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing an amplification product obtained by performing PCR amplification selectively on the rheumatoid related HLA-DRB1 allele (* 01, * 04 type) using the specific primer set of the present invention. As shown in FIG. 1, PCR amplification using the specific primer set of the present invention can detect rheumatism-related HLA-DRB1 alleles as primary two-digit types of * 01 and * 04 .
FIG. 2 is a photograph of an amplification product obtained by performing PCR amplification of rheumatoid related HLA-DRB1 allele (* 09, * 10 type) selectively using the specific primer set of the present invention and electrophoresis. As shown in FIG. 2, PCR amplification using the specific primer set of the present invention can detect the rheumatoid-related HLA-DRB1 allele primarily in two digits of * 09 and * 10 .
Figure 3 shows a primer set for amplifying other HLA-DRB1 alleles (* 03, * 07, * 08, * 11, * 12, * 13, * 14, * 15, * 16) together with the specific primer set of the present invention. PCR amplification was carried out using the PCR product and electrophoresis of the obtained amplification product. As shown in FIG. 3, when the PCR amplification is performed using the primer sets described above, it is possible to confirm whether the sample gene is amplified for each primer set, and whether the HLA-DRB1 allele of the amplified sample gene is homozygous This type change can be confirmed as low-resolution (two-digit type).
FIG. 4 shows a layout of a microarray DNA chip on which probes for HLA typing specific to rheumatism according to an embodiment of the present invention are integrated.
5 shows the PCR amplification products of various samples having eight rheumatoid related HLA-DRB1 alleles (* 0101, * 0401, * 0404, * 0405, * 0408, * 0410, * 0901, * 1001) It is a fluorescence image photograph of DNA chip confirmed after hybridization to microarray DNA chip.

Hereinafter, the present invention will be described in more detail based on examples. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Example 1: Preparation of primer

In order to analyze HLA-DRB1 genotypes, HLA-DRB1 gene homology analysis was performed. For this purpose, the sequence information was referenced to the IMGT / HLA database and the exon 2 nucleotide sequence homology of the HLA-DRB1 gene (* 01, * 04, * 09, * 10 types) of the allele genes related to rheumatism among the alleles of the genes * 01 to * 16 were selected and designed as forward primers capable of selectively amplifying only the HLA- DRB1 allele .

In addition, the reverse primer was designed by selecting conserved sequences for all HLA-DRB1 alleles. Such a primer set of the present invention can selectively amplify allelic genes associated with rheumatism to eliminate ambiguity due to other allelic gene amplification products.

In the present embodiment to be described later, the following primers are used. These primers were synthesized by asking Bioni Company of Korea (Daejeon Metropolitan City).

Primers for PCR amplification for the HLA-DRB1 allele (* 01, * 04, * 09, * 10 type)

Forward primer

DE2F-01 (* 01, codon no. 7 to 14): 5'-TTCTTGTGGCAGCTTAAGTTTGA-3 '(SEQ ID NO: 11)

DE2F-04 (* 04, codon no. 6 to 13): 5'-CGTTTCTTGGAGCAGGTTAAACAT-3 '(SEQ ID NO: 12)

DE2F-09 (* 09, codon no. 6 to 13): 5'-CGTTTCTTGAAGCAGGATAAGTTT-3 '(SEQ ID NO: 13)

DE2F-10 (* 10, codon no. 6 to 13): 5'-CGTTTCTTGGAGGAGGTTAAGTTT-3 '(SEQ ID NO: 14)

Reverse primer

DE2R-2 (consensus, codon no. 95 to 89): 5'-CTCGCCGCTGCACYGTGAA-3 '(SEQ ID NO: 15)

In addition, the forward primer of SEQ ID NO: 11 to SEQ ID NO: 14 and the reverse primer of SEQ ID NO: 15 used for the symmetric or asymmetric polymerase chain reaction (PCR) are subjected to a hybridization reaction, (Cytochrome c), cy3, or cy5, or biotin attached thereto. When biotin was attached thereto, it was used to bind to streptavidin-cyanine after the hybridization reaction. A preferred example is to attach cy3 to the primer end.

Example 2: Performing PCR amplification reactions on DNA samples of rheumatoid patient group and normal group

DNA samples were collected from Hanyang University Rheumatology Hospital, which constructed a large-scale clinical cohort (1,900 rheumatic patients and 1,130 normal patients) by accumulating DNA samples from Korean rheumatoid arthritis patients and systematically databaseing clinical information for many years. DNA can also be directly extracted from blood samples from patients with rheumatoid arthritis using the DNeasy blood & tissue kit from QIAGEN for PCR amplification.

In order to selectively amplify only the rheumatoid related HLA-DRB1 allele, PCR amplification was performed using the primers prepared in Example 1 above. As a composition for amplification, a 2X Hot-Taq Master Mix (including PCR buffer, dNTP, and DNA polymerase) of JEES Biotech was used, and each forward primer had a final concentration of 250 nM , The reverse primer was added to a final concentration of 500 nM, and 50 ng of DNA template was added.

The PCR amplification temperature conditions were 95 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds after denaturation at 95 ° C for 10 minutes, and then kept at 4 ° C until the electrophoresis experiment was performed.

2 μl of gel loading buffer (0.2% orange G, 0.25% xylene cyanol FF, 60% glycerol) was added to 3 μl of the PCR amplification product under the above conditions and 1 μg / ml of ethidium bromide and the band of the PCR amplification product was confirmed in an image analyzer (HITACHI, Japan) equipped with a UV transilluminator after electrophoresis on a 1.5% agarose gel containing a bromide.

As shown in FIGS. 1 and 2, using the primer set of the present invention, it was possible to selectively amplify each type of HLA-DRB1 allele * 01, * 04, * 09, * 10 related to rheumatism, It was confirmed that cross-reactivity with the allelic gene did not occur. From these results, PCR amplification using the specific primer set of the present invention revealed that the HLA-DRB1 allele associated with rheumatism was primarily detected as a two-digit type of * 01, * 04, * 09 and * 10 It can be seen that

On the other hand, amplification of other HLA-DRB1 alleles (* 03, * 07, * 08, * 11, * 12, * 13, * 14, * 15, * 16) with the specific primer set of the present invention of Example 1 (Non-specific primer set) was used to perform additional PCR amplification under the conditions described above. The following primers are used as the non-specific primer sets used additionally. The following non-specific primers are all forward primers and the reverse primers of SEQ ID NO: 15 are commonly used. These primers were synthesized by asking Bioni Company of Korea (Daejeon Metropolitan City).

A primer set for amplification of the HLA-DRB1 allele (* 03, * 07, * 08, * 11, * 12, * 13, * 14, * 15, * 16)

DE2F-03,11,13,14: 5'-CGTTTCTTGGAGTACTCTACGTC-3 '(SEQ ID NO: 16)

DE2F-07: 5'-TTTCCTGTGGCAGGGTAAGTATA-3 '(SEQ ID NO: 17)

DE2F-08,12: 5'-TTTCTTGGAGTACTCTACGGG-3 '(SEQ ID NO: 18)

DE2F-15,16: 5'-CTGTGGCAGCCTAAGAGG-3 '(SEQ ID NO: 19)

PCR amplification was performed on the sample genes having various HLA-DRB1 genotypes using the above two types of primer sets, and the amplification method and amplification conditions were PCR amplification performed using only the primer set of SEQ ID NOS: 11 to 15 Reaction was the same as in the case of the reaction. The resulting amplification product of each of the two types of primer sets was subjected to electrophoresis.

As shown in FIG. 3, PCR amplification was performed using the above-mentioned two types of primer sets to confirm whether the sample gene was amplified for each of the primer sets. Thus, the presence or absence of the HLA-DRB1 allele related to rheumatism It was possible to distinguish it by low resolution of two digits. In addition, the HLA-DRB1 allele of the amplified sample gene could be confirmed as homozygous mutation and heterozygous mutation as low-resolution (two-position type).

In other words, in the result of the electrophoresis of FIG. 3, when the PCR reaction was carried out using the specific primer set of the present invention, alleles of the four-digit subtypes belonging to the type * 04 were identified in the left lane as a dark band , And no band was observed in the right lane when the PCR reaction was performed using a non-specific primer set. Therefore, from this electrophoresis result, the HLA-DRB1 allele of the sample was firstly judged to be homozygous * 04 type (low resolution result).

On the other hand, for the samples having the HLA-DRB1 allele genotype of * 0408 / * 0701, bands were observed not only in the left lane but also in the right lane, and for the samples having the HLA-DRB1 allele of * 0408 / A weak band was observed in the right lane and the HLA-DRB1 alleles of each of the samples were firstly judged to be heterozygotes of the types * 04 / * 07 and * 04 / * 12 (low resolution results) I could.

Example 3: Preparation of a probe for HLA typing specific to rheumatism

In this Example, primers for HLA typing specific to rheumatism were designed and manufactured based on homology analysis of IMGT / HLA database. Sequence information of the probe for the hybridization reaction is shown in Table 1 below. Probes for HLA typing specific to the rheumatism of the present invention include one positive control probe, one probe for identifying types * 01, * 09, and * 10, and six probes for identifying type * 04 do.

These probes were prepared according to a method for synthesizing oligonucleotide probes generally known in the art or were commissioned by an institute specialized in oligonucleotide synthesis (for example, Bioni Company, Daejeon, Korea). On the substrate coated with aldehyde Amino link was attached to the 5 'end of all probes to covalently bind the oligonucleotides. Nine oligo (dT) was added to minimize spatial interference between the probes integrated on the slide glass during the hybridization reaction. The probes used in the examples were completed.

Table 1: Probe sequences for analyzing HLA-DRB1 genotypes related to rheumatism

0 "or" 1 "for each probe, depending on whether the PCR amplification product of the sample DNA of the rheumatic patient group and the normal group and the probes of Table 1 are hybridized, as described in the following hybridization reaction example Binary codes can be generated by assigning a binary value, and thereby it is possible to specifically discriminate alleles of HLA-DRB1 related to rheumatism.

That is, since there are many mutations in the race and individual, the eight alleles of HLA-DRB1 alleles (* 0101, * 0401, * 0404, * 0405, * 0408, * 0410, * 0901, * 1001) (* 0101, * 0401, * 0404, * 0405, * 0408, * 0405, * 0408, and * * 0410, * 0901, * 1001), and the allele genotype of HLA-DRB1 can be specifically analyzed according to the signal pattern from these probe combinations.

Specifically, when a target amplification gene (fluorescently labeled) is hybridized to the probes, a value of "1" is assigned to the probe when the measured fluorescence intensity is equal to or higher than a constant cutoff value, and "0"" Based on these assigned values, a binary code consisting of "1" and "0" can be generated for the entire probe combination set, and each binary code thus generated can be used as a conflict between the HLA- Matching with the gene not only facilitates the identification of the HLA-DRB1 allele, but also enables analysis of the HLA-DRB1 allele pattern using a computer program when analyzing a large number of samples.

Example 4: Fabrication of microarray chip

Fabrication of the microarrayed DNA chip is performed using methods well known to those skilled in the art.

First, an aminolink was modified at the 5'-end of an oligonucleotide having a probe base sequence of Example 3 on a glass having an aldehyde functional group, and then the spatial interference between the probes integrated on the slide glass during the hybridization reaction 9 < / RTI > oligos (dT) were added to minimize probes to complete the probes used in the examples of the present invention.

In order to prepare the microarray chip in which the oligonucleotide probes were immobilized, the amino link was immobilized on a CSS-100 silylated slide (Cel Associate, USA) at a concentration of 50 μM. The same amount of 3X SSC solution as that of the probe was mixed and collected and reacted at room temperature for 12 hours or more. After completion of the reaction, the slides were washed once with 0.1% SDS for 5 minutes and twice with distilled water for 5 minutes. The prepared chip was reacted with sodium borohydride solution (0.625 g NaBH ₄ , 168.75 ml ddH ₂ O, 18.75 ml PBS, 62.5 ml of 100% EtOH) for 5 minutes, then washed twice with distilled water for 5 minutes, ≪ / RTI > for 5 minutes.

The prepared microarray chip was divided into four sub-arrays. The microarray chip prepared in the hybridization experiment was covered with a CoverWell perfusion chamber (Grace-Bio Labs, USA) so that the microarray chip could react independently. The layout (layout) of the microarray chip in which the probes (SEQ ID NO: 1 to SEQ ID NO: 10) for analyzing the rheumatoid-related HLA-DRB1 genotype in Table 1 are integrated is shown in FIG.

Example 5: Hybridization reaction

PCR amplification products amplified in accordance with Example 1 and Example 2 and bound to a labeling substance (for example, cy3) were mixed at a ratio of 1: 9 with a hybridization solution (3X SSC, 0.3% Sarcosyl). The thus prepared hybridization solution was reacted with the microarray chip of FIG.

That is, the hybridization solution prepared as described above was applied to a chamber containing the microarray chip of FIG. 4 prepared in Example 4 and reacted at 60 ° C for 1 hour. Then, the cells were firstly washed in 1X SSC, 0.1% SDS solution for 5 minutes and again in 1X SSC solution for 5 minutes. Thereafter, it was further washed in a 0.1X SSC solution for 5 minutes. The washed slide was dried in a centrifuge at a speed of 800 rpm for 5 minutes.

In order to confirm the hybridization result of the microarray chip of FIG. 4, the hybridization signal (fluorescence signal) of the microarray chip that has been reacted is scanned using GenePix 4000B (Axon Instrument, USA) And the positive and negative of each probe were judged to be fluorescence values. Fluorescence intensity data was analyzed using GenePix Pro 4.1 software (Axon instrument, USA) and local background values were subtracted from the mean of each spot.

In the course of fluorescence data analysis, the cut-off value of each probe was set using the UCLA DNA panel and the sample DNA provided first in the course of the microarray chip experiment analysis, and the negative / positive was determined based on this. The negative / positive patterns according to each HLA-DRB1 allele were confirmed to be consistent with the known sequence of HLA-DRB1 allele, and 0 for negative and 1 for positive / Positive test was conducted. The results were confirmed by matching the HLA-DRB1 allele pattern matching with the hybridization experiment result of the microarray chip by preparing the expected positive / negative table.

On the basis of this basic experimental result, in this embodiment, when the amplified gene (fluorescence-labeled) to be a target is hybridized to the probes, if the fluorescence intensity measured is equal to or greater than a constant cutoff value, A value of "0" is assigned if the value is less than the cutoff value, and a binary code consisting of "1" and "0 & See Table 2).

Table 2: Correspondence table of binary code and HLA-DRB1 allele according to microarray chip result

As shown in Table 2, when the generated binary codes are matched with the allele of HLA-DRB1 to be analyzed, it is easy to identify HLA-DRB1 alleles. In addition, when a large amount of samples are analyzed, HLA-DRB1 allele genotype pattern analysis is possible.

In addition, in the allele genotype analysis of HLA-DRB1, allele 1 and allele 2 present on the diploid phase can be represented by a combination of the respective binary codes generated for each of them, so that homozygotes or heterozygotes Analysis of the heterozygote can be easily performed by a combination of binary codes.

In this regard, FIG. 5 shows the results of PCR amplification of various samples having eight HLA-DRB1 alleles (* 0101, * 0401, * 0404, * 0405, * 0408, * 0410, * 0901, * 1001) Of the present invention is hybridized to the microarray DNA chip of the present invention and confirmed.

5 shows that the fluorescence image data of the DNA chip of FIG. 5 according to the HLA-DRB1 allele genotype and the binary code data of Table 2 correspond to each other. Thus, the present invention provides a method for detecting rheumatoid related HLA- DRB1 allele genotype in rheumatism-related HLA-DRB1 allele genotypes can be easily and quickly and accurately analyzed while high-resolution analysis is possible.

For example, in the case of the HLA-DRB1 * 0404 / * 0408 heterotypes, the binary code may be represented as a heterozygote code of 1001010100/1001100000. The fluorescence pattern corresponding to the presence or absence of a fluorescent spot corresponding to the values 0 and 1 And the fluorescence pattern on the microarray chip of FIG. 5 (the first fluorescence pattern at the lower end) coincide with each other.

In the case of the HLA-DRB1 * 0101 / * 0410 heterotypes, the binary code can be displayed as a heterozygote code of 1101100100/1010010100. The fluorescence patterns corresponding to the presence or absence of fluorescent spots corresponding to the values of 0 and 1 And the fluorescent pattern on the microarray chip of 5 (the second lower fluorescent pattern) coincided with each other.

In addition, in the case of HLA-DRB1 * 0401 / * 0901 heterotypes, a binary code can be represented as a heterozygote code of 1001100100/1000101010. The fluorescence pattern corresponding to the presence or absence of fluorescent spots corresponding to the values 0 and 1 It was confirmed that the fluorescence pattern on the microarray chip of FIG. 5 (third lower fluorescence pattern) coincided.

As described above, the probe composition for HLA typing specific to rheumatism of the present invention is capable of inspecting a large amount of sample in a short time while greatly shortening the time for analyzing the sample compared with the conventional method, It is possible to perform allelic genotyping analysis at high resolution, so that it is possible to accurately and effectively diagnose and predict the onset and risk of rheumatism-related diseases at a genetic level.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

<110> Industry-University Cooperation Foundation Hanyang University          EONE REFERENCE LABORATORY <120> Probes, DNA chip and kit for HLA typing specific to rheumatism,          and method for genotyping HLA alleles specific to rheumatism          using the same <130> PN130625 <160> 19 <170> Kopatentin 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe DRB1-PC <400> 1 gacagcgacg tgggggag 18 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Probe DRB1-01-1a <400> 2 ttgctggaaa gatgcatcta taaccaa 27 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe DRB1-04-1a <400> 3 gcggcctagc gccgagtac 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe DRB1-04-1-1a <400> 4 gcggcctgat gccgagtac 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe DRB1-04-1-2b <400> 5 ggcggcctga cgctgagtac 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe DE2-04-1-3b <400> 6 aactacgggg ttgtggaga 19 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Probe DE2-04-1-5a <400> 7 gggccgaggt ggaca 15 <210> 8 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Probe DE2-04-1-6 <400> 8 ggccgcggtg gaca 14 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe DRB1-09-2b <400> 9 cggtatctgc acagaggcat ctat 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe DRB1-10-1 <400> 10 ctggaaagac gcgtccataa ccaa 24 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer DE2F-01 <400> 11 ttcttgtggc agcttaagtt tga 23 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer DE2F-04 <400> 12 cgtttcttgg agcaggttaa acat 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer DE2F-09 <400> 13 cgtttcttga agcaggataa gttt 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer DE2F-10 <400> 14 cgtttcttgg aggaggttaa gttt 24 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer DE2R-2 <400> 15 ctcgccgctg cacygtgaa 19 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer DE2F-03,11,13,14 <400> 16 cgtttcttgg agtactctac gtc 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer DE2F-07 <400> 17 tttcctgtgg cagggtaagt ata 23 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer DE2F-08,12 <400> 18 tttcttggag tactctacgg g 21 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer DE2F-15,16 <400> 19 ctgtggcagc ctaagagg 18

Claims (9)

In a probe composition for HLA typing specific for rheumatism,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 4 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 6 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 8 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
10. A probe composition for HLA typing specific to rheumatism comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 10 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide. In a DNA chip for HLA typing specific for rheumatism,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 4 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 6 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 8 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide,
10. A DNA chip for typing specific for rheumatism comprising at least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 10 and an oligonucleotide having a nucleotide sequence complementary to the oligonucleotide. 3. The method of claim 2,
Wherein the probes are microarrayed on the same substrate. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; DRB1 allele comprising a primer set consisting of the forward primers of SEQ ID NOs: 11 to 14 and the reverse primer of SEQ ID NO: 15, the DNA polymerase, the dNTPs, the PCR buffer solution and the PCR amplification product-specific HLA-DRB1 allele . &Lt; / RTI &gt; Amplifying the sample DNA by performing PCR,
Hybridizing said PCR amplification product to probes integrated on a DNA chip for HLA typing specific to the rheumatism defined in claim 2;
And analyzing the HLA-DRB1 allele according to the hybridization status. 6. The method of claim 5,
In the analysis of the HLA-DRB1 allele genotype, the rheumatoid-related HLA-DRB1 allele is discriminated to a subtype of 4-digit number. The method according to claim 5 or 6,
The step of analyzing the HLA-DRB1 allelotype comprises:
0 "for a probe that assigns a value of" 1 " for a probe whose fluorescence intensity measured above the cutoff value is greater than the cutoff value after hybridization of the fluorescently labeled PCR amplification product to the probes and which exhibits fluorescence intensity below the cutoff value, Assigning a value of &lt; RTI ID = 0.0 &gt;
Generating a binary code "1" and "0" for the entire probe combination set based on the assigned value;
And comparing each of the generated binary codes with an allele of HLA-DRB1 to be analyzed, thereby analyzing the rheumatism-specific HLA-DRB1 allele genotype. 8. The method of claim 7,
A combination of binary codes of allele 1 and allele 2 present on diploid is generated and the HLA-DRB1 allelic genotype is analyzed as a homozygote or a heterozygote from a combination of these binary codes Wherein the HLA-DRB1 allele is selected from the group consisting of HLA-DRB1 and HLA-DRB1 alleles. A DNA chip defined in claim 2 or 3,
A primer set consisting of the forward primers of SEQ ID NOS: 11 to 14 and the reverse primer of SEQ ID NO: 15 for specifically amplifying the rheumatoid related HLA-DRB1 allele,
And a labeling means for detecting an amplified gene product hybridized with the DNA chip, wherein the HLA-DRB1 allele genotype is capable of analyzing rheumatism-specific HLA-DRB1 allele.

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