KR20190079547A - DNA Typing Kits and Diagnostic Kits for Detecting Cancer using Minisatellites of RB1 Gene - Google Patents

Abstract

The present invention relates to a DNA typing kit and cancer diagnostic kit using of polymorphic minisatellites of retinoblastoma 1 (RB1) gene, and various applications thereof. According to the present invention, polymorphic RB1-minisatellites 1 (MS1) in the RB1 gene are transferred through reduction division in accordance with Mendelian inheritance such that DNA typing thereof is performed, thereby being effectively used for a paternity test, a blood relation test, or a forensic appraisal of an entity. Moreover, when an allelomorphic character repeated 23 or 24 times in a polymorphic RB1-MS1 region is included, susceptibility to a cancer is more highly represented than that of a normal person such that the polymorphic RB1-MS1 can be used as important data in estimation diagnosis as an important marker for investigating the susceptibility to cancer, thereby being useful in related industries.

Description

[0001] The present invention relates to a DNA typing kit and a kit for cancer diagnosis using the polymorphic sub-satellite of the RB1 gene,

The present invention relates to a DNA typing kit and a cancer diagnostic kit using the polymorphic bovine satellite of the RB1 gene and its various applications.

The RB1 (Retinoblastoma 1) gene was first discovered in a disease called retinoblastoma, and acts as a suppressor gene that suppresses tumors under normal conditions, but inhibits the production of functional proteins when mutations occur This can not control cell division, and cells can not control the formation of malignant tumors, resulting in the division of the phenomenon occurs such as cancer.

When RB1 was compared with normal tissues, it was confirmed that expression of RB1 was relatively low in colorectal cancer and prostate cancer. In addition, changes in the RB1 gene have been reported in lung cancer, osteosarcoma, and melanoma, and the low expression or mutation of this gene inactivates protein RB1, thus contributing to tumor formation.

The repeated sequence, which accounts for about 45% of the human genome, plays an important role in understanding the evolution of the entire genome, including the generation of new genes and variations in diversity, It is highly related to incurable diseases. Among the various repeat sequences, the tandem repeat (TR) sequence occupies more than 10% of the human genome, and the minisatellite (MS) belonging to the sequence consists mainly of repeating units of 10 to 100 bp , And the length varies from several hundred bp to about 20 kb or more depending on the number of repetitions. This repetitive sequence is mainly present in the gene regulatory region and the intron region and affects the gene expression, but it exists in the exon portion like the Mucin gene and may directly affect the function of the gene. Mutations in these regions are highly associated with many diseases and play an important role in the regulation of gene expression and can be used as diagnostic markers. Therefore, TR analysis and confirmation of polymorphisms in the RB1 gene could lead to a study on the relationship with cancer, thus completing the present invention.

The object of the present invention is to provide a personal identification microsatellite RB1-MS1 (minisatellite).

It is also an object of the present invention to provide a primer set composition for detecting polymorphonuclear RB1-MS1.

It is also an object of the present invention to provide a DNA typing kit for detecting polymorphonuclear RB1-MS1.

It is also an object of the present invention to provide a DNA typing method.

It is also an object of the present invention to provide a cancer diagnostic composition.

It is also an object of the present invention to provide a cancer diagnostic kit.

It is another object of the present invention to provide an information providing method for diagnosing cancer susceptibility.

In order to accomplish the above object, the present invention provides a method for identifying a personally identifiable polymorphic small satellite RB1-MS1 (minisatellite) represented by a nucleotide sequence from positions 123368 to 124744 of the RB1 (Retinoblastoma 1) gene (Genbank No. NC_000013.11) to provide.

In addition, the present invention provides a primer set composition for detecting polymorphonuclear RB1-MS1 comprising the nucleotide sequence shown in SEQ ID NOs: 2 and 3.

In addition, the present invention provides a DNA typing kit for detecting polymorphonuclear RB1-MS1 comprising the above composition.

Also, the present invention provides a method for preparing a recombinant vector comprising the steps of: (a) performing PCR using genomic DNA isolated from a specimen as a template and using the kit described above; And (b) identifying the polymorphonuclear RB1-MS1 by separating the PCR product by electrophoresis; The method comprising the steps of:

The present invention also provides a cancer diagnostic composition comprising a primer set for detecting a polymorphonuclear RB1-MS comprising the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3.

The present invention also provides a cancer diagnostic kit comprising a primer set for detecting a polymorphonuclear RB1-MS1 comprising the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3.

Also, the present invention provides a method for producing a recombinant DNA comprising the steps of: (a) performing PCR using genomic DNA isolated from a specimen as a template and using the kit; And (b) separating and identifying the PCR product by electrophoresis to analyze the length of the allelic variant of the polymorphonuclear RB1-MS1. The present invention also provides an information providing method for cancer susceptibility diagnosis.

Since the polymorphonuclear RB1-MS1 (minisatellite) in the RB1 (Retinoblastoma 1) gene of the present invention is transmitted through meiosis according to the Mendelian genetic makeup, DNA typing can be used effectively to confirm parental identification, . In addition, the RB1-MS1 polymorphic so-called RB1-MS1 polymorphism has an important role in investigating the susceptibility to cancer, as the susceptibility of the RB1-MS1 polymorphic sub- Markers that can be used as useful data for predictive diagnosis can be useful for related industries.

Fig. 1 is a schematic diagram showing the position and orientation of the RB1 gene located at the chromosome end of human 13; Fig. (A) is the result of analyzing the band structure of chromosome 13 by the MAP Viewer of the NCBI website. (B) is a schematic representation of the RB1 gene on chromosome 13q14.2.
2 is a schematic diagram showing the structure of the RB1 gene and the position of the minisatellite (MS) in the gene. The exon is indicated by a black line, and the location of the minisatellite detected by the Tandem Repeats Finder Program is indicated by an asterisk (*).
Figure 3 is a graph showing the results of electrophoresis showing polymorphism of RB1-MS1 in a female control group not associated with cancer.
Figure 4 shows the results of electrophoresis showing polymorphism of RB1- MS1 in cancer-free male controls.
Figure 5 shows the results showing that the RB1-MS1 region is genetically transferred between parent and offspring. Lane 1 is the grandfather, lane 2 is the grandmother, lane 3 is the father, 4 is the mother, and the DNA samples of the offspring are shown as lane 5 and lane 6, respectively.
6 is a graph showing the results of electrophoresis showing polymorphism of RB1-MS1 in breast cancer patients.
7 is a graph showing the results of electrophoresis showing polymorphism of RB1-MS1 in bladder cancer patients.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed description of known techniques well known to those skilled in the art may be omitted. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. Also, terminologies used herein are terms used to properly represent preferred embodiments of the present invention, which may vary depending on the user, intent of the operator, or custom in the field to which the present invention belongs.

Therefore, the definition of these terms should be based on the contents throughout this specification. Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise.

The present invention provides a personally identifiable polymorphic small satellite RB1-MS1 (minisatellite) represented by a nucleotide sequence of the 123368th to 124744th nucleotides of the RB1 (Retinoblastoma 1) gene (Genbank No. NC_000013.11).

The term "minisatellite " in the present invention means a sequence in which a basic unit consisting of about 10 to 100 bp nucleotides is repeated several times in the same manner. Such a structure is called" tandem repeat sequences " "

The term "polymorphism " in the present invention means a case where two or more alleles exist in one locus.

In the present invention, the term "polymorphic minisatellites" means that the bovine satellite corresponding to the chain repeat sequence has two or more alleles in all people and has a polymorphism that differs from person to person.

The term "allele " in the present invention refers to various types of genes that exist on the same locus of a homologous chromosome.

The polymorphic small satellite RB1-MS1 may be represented by the nucleotide sequence of SEQ ID NO: 1, and the polymorphic so called may represent a tandem repeat in the intron 17 region.

In order to identify polymorphonuclear satellites that can be used as an individual identification marker, the present inventors focused on the RB1 gene having the above characteristics and analyzed its gene structural characteristics using BLAST. As a result, one gene It first identified the presence of satellites and named it "RB1-MS1". Further, the present inventors confirmed that the polymorphonuclear RB1-MS1 was transferred through meiosis according to the Mendelian genetic make-up.

In addition, the present invention provides a primer set composition for detecting polymorphonuclear RB1-MS1 comprising the nucleotide sequence shown in SEQ ID NOs: 2 and 3.

In the present invention, the term "primer " refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The appropriate length of the primer may vary depending on the purpose of use, but is generally comprised of 15 to 30 bases. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template.

In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or It may contain an intercalating agent and is a forward and reverse primer designed to specifically detect or diagnose polymorphonuclear RB1-MS1 for the purposes of the present invention.

The primer set of the present invention can be chemically synthesized using a phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with an analogue, and modification between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In addition, the present invention provides a DNA typing kit for detecting polymorphonuclear RB1-MS1 comprising the above composition.

The kit may include one or more other component compositions, solutions or devices suitable for the assay method as well as a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 2 and 3.

Also, the present invention provides a method for preparing a recombinant vector comprising the steps of: (a) performing PCR using genomic DNA isolated from a specimen as a template and using the kit described above; And (b) identifying the polymorphonuclear RB1-MS1 by separating the PCR product by electrophoresis; The method comprising the steps of:

In the DNA typing method of the present invention, step (a) comprises amplifying the target sequence (polymorphic satellite RB1-MS1 of SEQ ID NO: 1) by performing PCR using the genomic DNA extracted from the sample as a template and using the primer set .

The specimen is a specimen for inspecting a subject. The specimen is selected from the group consisting of cells, tissues (biopsy samples), blood, hair, saliva, epidermis, semen, quality samples, separated cells, tissue samples, dandruff and ashes But are not limited to, but not limited to, species.

In one embodiment of the present invention, it was confirmed that regions 25 and 27 of the RB1 gene polymorphism subunit genetically transmitted through parental and progeny meiosis. This confirms that the bovine satellites in the RB1 gene are inherited by Mendel's law and are used as efficient markers for paternity or forensic emotions (Example 4, FIG. 5).

In the DNA typing method of the present invention, the step (b) is a step of detecting the polymorphonuclear RB1-MS1 of SEQ ID NO: 1 by separating the PCR amplification product obtained through the above process by electrophoresis.

To facilitate identification of PCR amplification products, PCR amplification products can be labeled with radioactive isotopes or fluorescence. The label may be obtained by attaching a fluorescent substance or an isotope to the 5 'end of the primer, or by adding a terminal deoxynucleotidyl transferase to the 3' end after PCR. If it is not labeled, it can be identified by silver staining after electrophoresis.

The PCR amplification product separated by electrophoresis was subjected to electrophoresis to measure the length of the DNA fragment amplified by PCR for detection of the polymorphonuclear RB1-MS1 of the present invention or mass spectrometry to measure the mass of the amplified fragment ), A method of measuring the difference of the base sequence by hybridization, and a method of directly determining the base sequence can be used. As the category of the test for measuring the complement, there may be included a DNA array in which a standard DNA known in advance such as a DNA chip is adhered to the surface of a substrate and then reacted with the DNA of the sample to identify the characteristics of the sample DNA .

In order to detect the polymorphonuclear RB1-MS1 of the present invention, a device for detecting a label attached to DNA of a sample or a DNA fragment can be detected. As a preferable detection method, there are autoradiography and scintillation counting methods of DNA labeled with radioactive isotope, and when labeled with fluorescence, ABI automatic base analyzer of Applied Biosystems or Hitachi ) FMBIO is used to detect fluorescence, and if there is no label, there is silver staining.

The DNA typing method can be used for paternity confirmation, blood donation confirmation, or forensic medical examination of an individual.

The present invention provides a cancer diagnostic composition comprising a primer set for detecting a polymorphonuclear RB1-MS comprising the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3.

The present invention also provides a cancer diagnostic kit comprising the composition.

Wherein the cancer is selected from the group consisting of breast cancer, bladder cancer, prostate cancer, cerebrospinal tumor, head and neck cancer, lung cancer, thymoma, mesothelioma, esophageal cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer, biliary cancer, kidney cancer, prostate cancer, But are not limited to, at least one selected from the group consisting of cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma and skin cancer, preferably breast cancer and / or bladder cancer.

The kit may include one or more other component compositions, solutions or devices suitable for the assay method as well as a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 2 and 3.

For example, the kit of the present invention may be a kit containing essential elements necessary for performing PCR. In addition to the above primer set, the PCR kit can also include enzymes such as test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNases, RNAse inhibitors, DEPC-water (DEPC-water) and sterilized water.

In addition, the kit may include a brochure. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The brochure may include instructions on the surface of a package that includes a brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure may include information that is disclosed or provided through an electronic medium, such as the Internet.

Also, the present invention provides a method for producing a recombinant DNA comprising the steps of: (a) performing PCR using genomic DNA isolated from a specimen as a template and using the kit; And (b) separating and identifying the PCR product by electrophoresis to analyze the length of the allelic variant of the polymorphonuclear RB1-MS1. The present invention also provides an information providing method for cancer susceptibility diagnosis.

If the repetition of the alleles of the polymorphonuclear RB1-MS1 is 23 or 24, it may be determined that the susceptibility to cancer is high, but the present invention is not limited thereto.

Wherein the cancer is selected from the group consisting of breast cancer, bladder cancer, prostate cancer, cerebrospinal tumor, head and neck cancer, lung cancer, thymoma, mesothelioma, esophageal cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer, biliary cancer, kidney cancer, prostate cancer, But are not limited to, at least one selected from the group consisting of cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma and skin cancer, preferably breast cancer and / or bladder cancer.

In an embodiment of the present invention, a haplotype pattern between a normal human and a breast cancer patient was analyzed to determine the relationship between the RB1 polymorphism and the breast cancer. As a result, the RB1 polymorphonuclear RB1-MS1 Confirming that the repetitive units of 53 bp appeared at positions 23, 24, 25, 26, 27 and 28, confirming the association between the RB1 polymorphonuclear RB1-MS1 and breast cancer (Example 5, Fig. 6) .

In addition, we confirmed that RB1-MS1 has a susceptibility to breast cancer of about 6.7-fold when it has a genotype consisting of alleles 25 and 27 and rare alleles of 23 and 24 lengths. In the case of RB1-MS1, 23, and 24 of the rare allelic pattern were found to increase in the control group not associated with cancer (Example 5, Table 7).

In addition, the correlation between RB1 polymorphism and bladder cancer was found to be higher in RB1-MS1 than in the non-cancer-related control group in bladder cancer patients with 23 and 24-long rare alleles. In other words, it was confirmed that the susceptibility to bladder cancer was increased by about 7.6 times (Example 6, Table 6) when a genotype consisting of the 25 alleles of RB1-MS1 and the 23 alleles of 24 alleles was obtained.

The target sequence amplified by the PCR may be labeled with a detectable labeling substance. In one specific example, the labeling material can be, but is not limited to, a material that emits fluorescence, phosphorescence, or radiation. Preferably, the labeling substance is Ethidium Bromide (EtBr), Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. In addition, if the radioactive isotope such as 32P or 35S is added to the PCR reaction solution, the amplification product may be synthesized and the radiation may be incorporated into the amplification product and the amplification product may be labeled with the radiation.

Detection of the amplified product can be performed through DNA chip, gel electrophoresis, radiation measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one method of detecting the amplification product, gel electrophoresis can be performed. Gel electrophoresis can be performed using agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then the radioactivity is measured by a radiation measuring device such as a Geiger counter or liquid scintillation counter The radiation can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

<Example 1> Structural analysis of RB1 gene

Structural features of RB1 (NC_000013.11 REGION: c1345070-1317744) gene were analyzed using BLAST. Specifically, in FIG. 1, the band structure of RB1 human 13 chromosome was analyzed by MAP Viewer of the NCBI website. In addition, the position numbers of the indices shown in Table 1 are shown in the NC_000013.11 REGION. Exon and intron regions present in the promoter region and the open reading frame (ORF) are examined, the positions of tandem repeats (TR) existing in the respective regions are determined, (MS) of about 10 ~ 100bp in sequence size. The results are shown in FIG. 1 and Table 1.

 As shown in Fig. 1 (A), the RB1 gene is located at the chromosome 13q14.2 of human chromosome 13, and the position and orientation of the RB1 gene are confirmed in Fig. 1 (B). As shown in Table 1, the RB1 gene has a size of about 178 Kb and is composed of 28 exons, and one chain repeat region is located in the 17th intron region.

SEQ ID NO / sequence Small satellite
(minisatellite) index
(indices) location Repeat size
(repeat size) Copies
(copy no.) Representative size
(bp) SEQ ID NO: 1 RB1- MS1 123368-124744 Int17 53 26 1,378 order agtcatcttc taccaaacct cacctccagc attggggagc acacttcaac acg

Example 2: Polymorphism analysis of the minisatellite (MS) of RB1 gene

In order to analyze the RB1 gene polymorphic satellite, one small satellite (minisatellite, MS, Table 1) in the RB1 gene was analyzed by PCR. The base sequences of the primers used in the PCR analysis are shown in Table 2 below. Specifically, allele patterns of four TRs present in the RB1 gene were compared using PCR using genomic DNA extracted from blood of 100 adult males and females. The genomic DNA was subjected to standard PCR conditions (50 mM Tris-HCl (pH 9.0), 50 mM MgCl ₂ , 0.2 mM dTTP, 0.2 mM dCTP, 0.2 mM dGTP and 0.2 mM dATP in a final volume of 40 μl) 3 &lt; / RTI &gt; primer.

PCR analysis of the DNA samples was carried out using 100 ng of genomic DNA as a template using an EmeraldAmp PCR Master Mix (Taq polymerase, TAKARA). The conditions of the cycle were 1 heat treatment for 2 minutes at 94 for RB1-MS1, 30 cycles of 94 to 30 seconds and 64.5 to 2 minutes, and the last extension step was extended at 72 for 10 minutes. For the RB1-MS1, the PCR product was injected into 2% SeaKem LE agarose gel and analyzed by electrophoresis (1 volt / cm) in TAE buffer.

As a result, one domain showed polymorphism and thus it was confirmed that it could be used as an individual identification marker.

Polymorphic satellite primer SEQ ID NO: Base sequence RB1- MS1 RB1- MS1 F SEQ ID NO: 2 tcttgtgaac taactgagcg aga RB1- MS1 R SEQ ID NO: 3 tggaagggac aaacattgaa acc

<Example 3> Analysis of RB1-MS1 (minisatellite 1) in female and male control groups not related to cancer

Using the method of Example 2 above, 468 female non-cancer control subjects were examined and alleles, their repeat units and patterns were analyzed. The results are shown in Table 3 and FIG.

Using the method of Example 2 above, 443 male controls not related to cancer were investigated, and alleles, their repeat units and patterns were analyzed. The results are shown in Table 6 and FIG.

As shown in Fig. 3 and Table 3, in the female control group not related to cancer, it was confirmed that the RB1-MS1 polymorphism satellites had 6 patterns and various bands appeared. Specifically, six alleles of about 1,382 bp, about 1,435 bp, about 1,488 bp, about 1,541 bp, about 1,594 bp, and about 1,647 bp were identified. Also, it was confirmed that the 53 bp repeating unit was repeated 24, 25, 26, 27, 28, and 29 times, indicating that the polymorphic satellite was confirmed.

In addition, as shown in Fig. 4 and Table 4, in the male control group not related to cancer, it was confirmed that the RB1-MS1 polymorphism satellites had 6 patterns and various bands appeared. Specifically, six alleles of about 852 bp, about 1,170 bp, 1,329 bp, 1,435 bp, about 1,488 bp and about 1,541 bp were identified. Also, it was confirmed that the repetitive units of 53 bp were repeated 14, 20, 23, 25, 26 and 27, and the polymorphic satellite was confirmed.

Repeat unit × Number of repetitions N = 936 ** frequency 53 × 24 One 0.001 53 × 25 690 0.737 53 × 26 26 0.028 53 × 27 216 0.231 53 × 28 2 0.002 53 × 29 One 0.001 ** "frequency = each N value / total N value", and if the value is less than 0.01, it corresponds to the rare polymorphism.

Repeat unit × Number of repetitions N = 886 ** frequency 53 × 14 One 0.001 53 × 20 One 0.001 53 x 23 One 0.001 53 × 24 0 0 53 × 25 646 0.729 53 × 26 27 0.030 53 × 27 210 0.237 ** "frequency = each N value / total N value", and if the value is less than 0.01, it corresponds to the rare polymorphism.

Example 4 Genetic Transmission Measurements of RB1 Polymorphic Small Satellites (RB1-MS1) by Meiosis

The DNA of the offspring is formed by exactly 50% from the father and mother. Thus, since the offspring's DNA has half of the father's DNA pattern and half of the mother's DNA pattern, the paternity identification genetic test will examine whether the DNA pattern of the father / mother shows the same DNA pattern on the offspring. Therefore, this region should contain a stable region that does not undergo recombination through meiosis. In this invention, genomic DNA is extracted from the blood of grandparents, parents, and offspring, and PCR is performed in the same manner as in Example 2, The allelic pattern of RB1-MS1 was confirmed. The results are shown in Fig.

As shown in FIG. 5, it was confirmed that regions 25 and 27 of the RB1 gene polymorphism so-called genetic precise transmission through parental and progeny meiosis. This indicates that bovine satellites in the RB1 gene are inherited by Mendel's law and can be used as effective markers for paternity or forensic emotions. Using these DNA typing markers, It is predictable.

Example 5: Measurement of the association between RB1 polymorphism (MS) and breast cancer

<5-1> Relationship between RB1 polymorphism and so-called breast cancer

To investigate the effect of the RB1 gene on gene expression of the RB1 gene in the present invention, 468 control subjects and 707 breast cancer patients who were not associated with cancer were analyzed for the RB1-MS1 region. Genomic DNA of normal and breast cancer patients And the PCR was performed in the same manner as in Example 2 to compare haplotype patterns between normal human and breast cancer patients. The results are shown in Table 5 and FIG.

As shown in Table 5 and FIG. 6, six alleles of about 1,329 bp, about 1,382 bp, about 1,435 bp, about 1,488 bp, about 1,541 bp and about 1,594 bp were found in the analysis of breast cancer patients, Were repeated 23, 24, 25, 26, 27 and 28, respectively.

Control group Breast cancer patient Repeat unit × Number of repetitions N = 936 Frequency N = 1414 Frequency 53 x 23 0 0 6 0.004 53 × 24 One 0.001 4 0.003 53 × 25 690 0.737 1,070 0.757 53 × 26 26 0.028 45 0.032 53 × 27 216 0.230 285 0.202 53 × 28 2 0.002 4 0.003 53 × 29 One 0.001 0 0

<5-2> Measurement of the association between alleles of RB1 polymorphism and breast cancer

A total of 11 alleles of RB1-MS1 were compared according to frequency by the common allele (1%) and the rare allele (1%). The results are shown in Table 6 below.

 As shown in Table 6, the patterns of RB1-MS1 having 23 or 24-long rare alleles in breast cancer patients were found to be more increased than those in the non-cancer-related control group. That is, the susceptibility of RB1-MS1 to allogeneic traits consisting of 25 and 27 alleles and 23 and 24 rare alleles showed a statistically significant increase (about 6.7 times) in breast cancer susceptibility OR = 6.70, 95% Cl: 0.85-52.52, P = 0.036 *).

These results indicate that RB1-MS1 is an important marker for susceptibility to breast cancer and can be used as an important data for prediction diagnosis.

heredity
characteristics Control group
468
[N (%)] Breast cancer patient
707
[N (%)] OR (95% CI) P OR (95% CI)
; P
TR23 / MR 23/25 0 3 (0.42) - 0.16 6.70
(0.85-52.52)
; 0.036 * 23/27 0 3 (0.42) - 0.16 24/25 1 (0.21) 3 (0.42) 1.99
(0.21-19.19) 0.54 24/27 0 1 (0.14) - 0.42 25/25 260 (55.6) 413 (58.42) 1.12
(0.89-1.42) 0.33 25/26 22 (4.70) 30 (4.24) 0.90
(0.51-1.58) 0.70 25/27 147 (31.4) 204 (28.85) 0.89
(0.69-1.14) 0.35 25/28 0 4 (5.66) - 0.36 26/26 0.42 - 26/27 - 13 27/27 1 (0.14) 32 27/28 0 0 - 27/29 4 (0.85) 13 (1.84) 2.17
(0.70-6.70) 0.17

Example 6 Measurement of Association Between RB1-MS1 Region and Bladder Cancer

PCR was performed in the same manner as in Example 1 to measure the relationship between RB1-MS1 and bladder cancer. Haplotype patterns between normal and bladder cancer patients were compared as shown in Table 6. A total of 10 alleles of RB1-MS1 were divided by frequency into the common allele (over 1% of occurrence) and the rare allele (case of less than 1% of occurrence) Respectively. The results are shown in Tables 7, 8 and 7.

As shown in Tables 7, 8 and 7, it was confirmed that RB1-MS1 had an increased pattern of the 23 and 24-long rare alleles in bladder cancer patients than in the control group not related to cancer. In other words, susceptibility to bladder cancer was increased about 7.6 times with a genotype consisting of 25 alleles of RB1-MS1 and 23 alleles of 24 alleles (statistical significance) (OR = 7.65, 95% Cl: 0.89-65.79, P = 0.029 *).

These results indicate that RB1-MS1 is an important marker for susceptibility to bladder cancer and can be used as an important data for predictive diagnosis.

Control group Bladder cancer patient Repeat unit × Number of repetitions N = 886 Frequency N = 588 Frequency 53 × 14 One 0.001 0 0 53 × 20 One 0.001 One 0.002 53 x 23 One 0.001 4 0.007 53 × 24 0 0 One 0.002 53 × 25 646 0.729 446 0.59 53 × 26 27 0.030 12 0.020 53 × 27 210 0.237 124 0.211

Genetic trait Control group Bladder cancer
patient OR (95% CI) P OR (95% CI)
; P 443 (%) 294 (%) 14/25 One 0 - 0.41 20/25 One One 1.51 (0.09-24.21) 0.77 23/25 One 4 6.10 (0.68-54.82) 0.07 7.65
(0.89-65.79)
; 0.029 * 24/25 0 One - 0.22 25/25 237 168 1.16 (0.86-1.56) 0.33 25/26 20 11 0.82 (0.39-1.74) 0.61 25/27 149 93 0.91 (0.67-1.25) 0.57 25/28 3 0 - 0.16 26/27 7 One 0.21 (0.03-1.74) 0.11 27/27 27 15 0.83 (0.43-1.59) 0.57

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> DNA Typing Kits and Diagnostic Kits for Detecting Cancer using          Minisatellites of RB1 Gene <130> PN1812-528 <150> KR 10-2017-0180610 <151> 2017-12-27 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> RB1-MS1 minisatellite <400> 1 agtcatcttc taccaaacct cacctccagc attggggagc acacttcaac acg 53 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > RB1-MS1 F_primer <400> 2 tcttgtgaac taactgagcg aga 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > RB1-MS1 R_primer <400> 3 tggaagggac aaacattgaa acc 23

Claims (14)

The RB1-MS (minisatellite) for identification of individuals identified as the nucleotide sequence from 123368 to 124744 of the RB1 (Retinoblastoma 1) gene (Genbank No. NC_000013.11). The method according to claim 1,
Wherein the polymorphonuclear RB1-MS1 is represented by the nucleotide sequence of SEQ ID NO: 1. The method according to claim 1,
Characterized in that the polymorphic so called tandem repeats in the intron 17 region. A primer set composition for detecting polymorphonuclear RB1-MS1 comprising the nucleotide sequence shown in SEQ ID NOs: 2 and 3. A DNA typing kit for detection of polymorphonuclear RB1-MS1 comprising the composition of claim 4. (a) carrying out PCR using the genomic DNA isolated from a specimen as a template and using the kit according to claim 5; And
(b) identifying the polymorphonuclear RB1-MS1 by separating the PCR product by electrophoresis; &Lt; / RTI &gt; The method according to claim 6,
Wherein the specimen is at least one selected from the group consisting of blood, hair, saliva, epidermis, semen, quality samples, separated cells, tissue samples, dandruff and ashes. The method according to claim 6,
Wherein the DNA typing method is used for paternity identification, blood donor identification, or forensic emotion of an individual. A primer set for detecting a polymorphonuclear RB1-MS1 comprising the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3. 10. The method of claim 9,
Wherein the cancer is selected from the group consisting of breast cancer, bladder cancer, prostate cancer, cerebrospinal fluid, head and neck cancer, lung cancer, thymoma, mesothelioma, esophageal cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer, Endometrial cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma and skin cancer. A cancer diagnostic kit comprising the composition of claim 10. (a) carrying out PCR using the genomic DNA isolated from a specimen as a template and using the kit of claim 11; And
(b) isolating and identifying the PCR product by electrophoresis and analyzing the length of alleles of the polymorphonuclear RB1-MS1. 13. The method of claim 12,
Wherein the cancer is selected from the group consisting of breast cancer, bladder cancer, prostate cancer, cerebrospinal fluid, head and neck cancer, lung cancer, thymoma, mesothelioma, esophageal cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer, Endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma and skin cancer. 13. The method of claim 12,
Further comprising the step of determining that the susceptibility to cancer development is high when the repetition of the alleles of the polymorphonuclear RB1-MS1 is 23 or 24 times the length.

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