KR101183084B1 - Minisatellites of MUC2 Gene and DNA Typing Kits and Diagnostic Kits for Cancer Using the Same - Google Patents

Abstract

The present invention relates to a polymorphic so called MUC2 gene and a DNA typing kit and a cancer diagnostic kit using the same. More specifically, the polymorphic so called MUC2 -MS7 in a MUC2 gene, a primer set for detecting the same, a DNA typing kit using the primer set. And cancer diagnostic kits.

Since MUC2- MS7 of the present invention is transmitted through meiosis according to the Mendelian heredity, DNA typing can be used for paternity, blood relative or forensic emotion of an individual, and amplifies the MUC2- MS7 region to generate rare alleles. The susceptibility to disease can be diagnosed by comparing the population frequency with normal individuals.

MUC2, polymorphic enantiomer, DNA typing, stomach cancer

Description

Minatellites of MUC2 Gene and DNA Typing Kits and Diagnostic Kits for Cancer Using the Same}

The present invention relates to a polymorphic so-called (minisatellite, MS) of the MUC2 gene secreted mucin, more specifically, polymorphic so-called MUC2- MS7 in the MUC2 gene, a primer set for detecting the same, using the primer set It relates to a DNA typing kit and a kit for cancer diagnosis.

Mucin is a major component of mucous substances that secretes mucous substances into respiratory organs such as epithelial cells and airways of the digestive organs, protecting the intestinal surface, the epithelial tissue from mechanical damage and chemical irritation of each organ. It acts as a lubricant in and digestive exercise. Twenty types of mucin genes that perform these functions have been identified to date, and according to the function, secretory mucins ( MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC9, MUC19 ) and membrane-bound mucins ( MUC1, MUC3A, MUC3B) , MUC4, MUC12, MUC17, MUC18, MUC20) .

Of these mucin genes, in particular, the secretory mucin MUC2, MUC5AC, MUC5B, MUC6 genes are all concentrated in the p15.5 region of the terminal region of human chromosome 11 to form a gene cluster (Fig. 1). It appears that the secretory mucin gene family is closely related to mutual gene expression and function.

Secretory mucin genes protect mucus and intestines by secreting mucus from different organs under normal conditions, but secretion or oversecretion occurs in organs that are unpredictable in the event of gene regulation or gene abnormalities. The abnormal phenomenon occurs. Due to this oversecretion, bronchial inflammation causes asthma or inflammatory diseases, and in the case of gastric cancer, it is reported that excessive mucus increases the tolerance to various pathogens, thereby increasing the frequency of tumors such as gastric cancer.

In addition, the secretory mucin constituting the gene cluster has been reported to cause instability because it contains a large number of repeat sequences in the central region of the gene because it is present in the gene terminal region. Structurally, secretory mucins were observed to have tandem repeats (TR) in the central region of the gene. Due to this TR structure, it exhibits a characteristic of viscous mucin, and structural variation occurs in the TR part as a cause of genetic diseases found in humans, and its meaning is very important. These highly complex mucin genes are not completely isolated from the current genome, but the nucleotide sequence is gradually revealed by the human genetic business.

Many recent studies have shown that these tandem repeat (TR) sequences make up more than 10% of the human genome, are responsible for many diseases, and are critical for the regulation and evolution of gene expression. TR sequences are divided into polymorphic ones that have only one length in every person and polymorphic ones that have two or more alleles that differ from person to person, depending on their length. Polymorphic repetition sequences have important significance as genomic markers that can be used for human genome mapping in early genome studies. Thus, TR analysis and polymorphism identification in structurally highly complex mucin genes can be used to study the association with disease.

Accordingly, the present inventors have diligently tried to develop a DNA typing kit and a diagnostic kit for gastric cancer using polymorphic polymorphism (minisatellite (MS)). As a result, the exon of MUC2 , a secretory mucin gene whose entire DNA sequence has been identified, has been identified. The polymorphic so called polymorphism (minisatellite (MS)) of MS7 in the region is transmitted through meiosis according to the Mendelian hereditary and confirmed that it is associated with susceptibility to cancer, and completed the present invention. In particular, the MUC2- MS7 region is located in the exon region, 69bp is a large and complex sequence repeated about 100 times, amplified by the PCR method for the first time in this experiment is significant.

One object of the present invention is to provide a MUC2- MS7 polymorphic minisatellites that can be used as personal identification markers and a primer set for detecting the polymorphic soot.

It is another object of the present invention to provide a DNA typing kit comprising the primer set useful for paternity identification, kinship identification or forensic evaluation of an individual, and a DNA typing method using the kit.

Still another object of the present invention is to provide a kit for diagnosing cancer, in particular gastric cancer, comprising the primer set, and a method for diagnosing cancer using the kit.

Genetic test I: DNA fingerprint test for personal identification

The present invention relates to a polymorphic variable number tandem repeat (VNTR) present in the exon region of the MUC2 gene. Since MUC2 -MS7 small satellite area present in the exon region of the gene MUC2 have a different number of repetitions for each individual corresponds to representing VNTR polymorphism. Accordingly, the present invention relates to a primer set for detecting polymorphism for the MUC2- MS7 region and to the diagnosis of DNA typing and mucin related diseases using the primer set.

(2) Genetic test II: Identification of paternity

The offspring's DNA is formed by inheriting exactly 50% of the father and mother. Therefore, the DNA of the offspring has half of the DNA pattern of the father and half of the DNA pattern of the mother. Therefore, the paternity genetic test examines whether the DNA pattern of the father / mother is the same DNA pattern for the offspring. Therefore, these regions should include stable regions where recombination does not occur through meiosis.

Since it was confirmed that the VNTR of the MUC2 gene found in the present invention is transmitted through meiosis according to the Mendelian heredity (see Example 2), it can be effectively used for paternity, kinship, or forensic emotion of an individual by DNA typing. have.

(3) Genetic Test III: MUC2  How to diagnose related diseases (e.g. stomach cancer)

We compared MUC2- MS7 so-called in normal and gastric cancer patients with more than 1% of normal population frequency and less than normal alleles and rare alleles and found that people with rare alleles were more likely to develop gastric cancer. Therefore, disease susceptibility can be diagnosed by amplifying the MUC2- MS7 region and comparing the frequency of the rare allele in normal and gastric cancer patients.

The present invention relates to a polymorphic so-called MUC2 -MS7 ( MUC2 -minisatellite7) for personal identification, consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the exon 31 region in the MUC2 gene.

The present invention also relates to a primer set for detecting polymorphic so-called MUC2- MS7. It is preferable that the primer set for polymorphic so-called MUC2- MS7 detection consists of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3.

The present invention also relates to a DNA typing kit for polymorphic so-called MUC2- MS7 detection comprising the primer set described above.

In addition, (1) using genomic DNA isolated from the tissue of the subject as a template, and performing PCR using the DNA typing kit; And (2) identifying the polymorphic so-called MUC2- MS7 by separating the PCR product by electrophoresis.

The DNA typing method is preferably used for paternity identification, kinship identification or forensic emotion of an individual.

In addition, the present invention comprises a primer set for detecting polymorphic so-called MUC2- MS7 consisting of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3; It relates to a kit for diagnosing cancer comprising DNA polymerase and dNTPs (dGTP, dCTP, dATP and dTTP). Here, the cancer preferably means gastric cancer.

In addition, (1) using genomic DNA isolated from the tissue of the subject as a template, and performing PCR using the cancer diagnostic kit; And (2) identifying the polymorphic so-called MUC2- MS7 by separating the PCR product by electrophoresis.

Hereinafter, the present invention will be described in detail.

Figure 1 (A) is the result of analyzing the band structure of human chromosome 11 by the MAP Viewer of NCBI website, Figure 1 (B) is a diagram showing the mucin gene on the 11p15.5 chromosome, MUC2, MUC5AC and MUC5B is towards the centromere, while MUC6 is towards the telomere. Figure 2 shows the structure of the MUC6 gene and the location of so-called (minisatellite, MS) in the gene. Exons were marked with a blue line, and the locations of the minisatellite detected by the Tandem Repeats Finder Program are marked with an asterisk (*).

According to the present invention, MUC2- MS7 present in the exon of the MUC2 gene can be used as a marker for personal identification, as it shows polymorphism. More specifically, MUC2- MS7 is located in the exon 31 region of the MUC2 gene, consisting of the nucleotide sequence shown in SEQ ID NO: 1, the repeat unit of 69bp repeats from 29 to 128 times depending on the individual.

Therefore, according to the present invention, it is possible to detect the polymorphic so-called by using a primer set capable of amplifying the MUC2- MS7 polymorphic so-called. Suitable primers are those capable of amplifying a portion of the MUC2- MS7 nucleic acid molecule and are based on nucleic acid sequences encoding at least 7 consecutive amino acids of the MUC2- MS7 nucleic acid. Generally, the primers have a length of 17-25 bp, preferably 20 bp, and at least about 60%, preferably at least about 75%, more preferably at least about 90% of the polynucleotides encoding MUC2- MS7. Has homology.

In one embodiment, it is preferable that the polymorphic so-called MUC2- MS7 detects the polymorphic so-called using a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 2 and 3.

Also, MUC2 -MS7 polymorphism so-called castle provides be passed through meiosis, in accordance with Mendel Ryan oil (also known as DNA typing or DNA testing, DNA profiling, Genetic fingerprinting ) The invention MUC2 -MS7 bovine DNA polymorphism typing using a satellite kit do. That is, the present invention provides a DNA typing kit comprising a primer set capable of detecting MUC2- MS7 described above. DNA typing refers to a technique for distinguishing between homologous individuals using DNA samples, in particular, by using a difference in the number of repeats of tandem repeats sequences.

In the present invention, the genomic DNA is isolated from an individual, and then the template is used. The polymorphic so-called can be identified by performing electrophoresis after PCR using the DNA typing kit, and confirming the number of repetitions of MUC2- MS7 on the gel. Here, isolation, PCR and electrophoresis of genomic DNA can all be carried out as known in the art. This DNA typing allows for the identification of individuals within the same family, so that paternity, blood, or related diseases can be diagnosed.

On the other hand, MUC2 of the present invention shows a high correlation with cancer, especially gastric cancer. Therefore, the susceptibility to cancer can be diagnosed by confirming the number of repetitions of MUC2- MS7. The rare allele of MUC2- MS7 was about 1.6 times higher in susceptibility to gastric cancer than normal. In addition, when comparing the rare alleles by length to the short rare alleles with 29-78 repetitions and the long rare alleles with 84-128 repetitions, MUC2 -MS7 was found to have gastric cancer with short repetitions. The sensitivity was about 2.09 times higher than that of the normal person.

Accordingly, the present invention provides a primer set for detecting polymorphic so-called MUC2- MS7, consisting of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3; It relates to a kit for diagnosing cancer comprising DNA polymerase and dNTPs (dGTP, dCTP, dATP and dTTP).

As described in detail above, since the polymorphic so-called MUC2- MS according to the present invention is transmitted through meiosis according to the Mendelian heredity, DNA typing may effectively be used for paternity identification, kinship identification or forensic emotion of an individual. We can diagnose the susceptibility to disease by amplifying the MUC2- MS7 region and comparing the frequency of the rare alleles with those of normal persons, and can use this to predict the probability of disease occurrence before the disease occurs. It can be used for preventive medicine. It can also be an important resource for the selection of the appropriate drug based on differences in each genotype.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention having ordinary skill in the art to which the present invention pertains. It will be obvious to him.

Example 1 Polymorphism Analysis of So-called Minisatellite (MS) Located at Exon 31 of MUC2 Gene

The location number of the indices shown in Table 1 shows the analysis of genomic DNA provided by the UCL site. MUC2 had a 31 th exon of about 9 Kb in size and a chain repeat region of about 7 Kb in the 31 exon region. The so-called information on the exon 31 region is shown in Table 1.

Chain repeat located in exon 31 region of MUC2 gene SEQ ID NO: So-called
(minisatellite) index
(indices) location order Repeat size
(repeat size) Representative copy number
(copy no.) Representative size
(bp) SEQ ID NO: 1 MUC2 -MS7 21278-26277 Exon31 CCAACCACGACACCCATCACCACCACCACTACGGTGACCCCAACCCCAACACCCACCGGCACACAGACC
(23 aa: PTTTPITTTTTVTPTPTPTGTQT) 69 104 7334

The polymorphic so called polymorphism (minisatellite, MS, Table 1) present in the MUC2 gene was analyzed by PCR. The base sequences of the primers used for PCR analysis are shown in Table 2 below.

Primers used to detect MUC2- MS7 polymorphism SEQ ID NO: So-called
(minisatellite) Primer Amino acid sequences SEQ ID NO: 2 MUC2 -MS7 MUC2 -MS7 F CATCACCACCACCACTACGGTGAC SEQ ID NO: 3 MUC2 -MS7 MUC2 -MS7 R CGGAGGATTGGATGTGGTCAACTC

Genomic DNA extracted from the blood of 100 adult men and women was used to compare the alleles of the MS7 (minisatellites1) site located in the exon 31 region in the MUC2 gene using PCR. As a result, it showed polymorphism and MUC2- MS7 region in exon showed polymorphism. Polymorphism in exon showed polymorphism, and it was previously unknown that the region having such a large and complex structure was amplified by PCR. will be.

Genomic DNA was purified under 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, final volume 40 μL ). Amplification was carried out using primers of 3.

PCR analysis of DNA samples was performed using COSMO GENETECH SP-Taq DNA polymerase (COSMO GENETECH, SP-Taq DNA polymerase) as a template with 100 ng of genomic DNA. The conditions of the cycle were heat treated once at 94 ° C. for 2 minutes, followed by 28 cycles of 45 seconds at 94 ° C., 15 seconds at 65 ° C., 5 minutes at 72 ° C., and then the last extension step was extended at 72 ° C. for 7 minutes. It was.

The PCR product was injected into 0.7% SeaKem LE agarose gel and analyzed by electrophoresis (1 volt / cm) in TAE buffer. GelStar staining reagent (LONZA) was diluted 1 × in TAE buffer. The electrophoretic gel was soaked in GelStar staining reagent and shaken for 30 minutes while covering the surface. As a result, all of them showed polymorphism so-called that it can be used as a personal identification marker. Therefore, below, the number of individuals was increased and further analysis was performed (Table 3, FIG. 3). In this case, N is the number of alleles, and each person has two alleles, which is twice the number of samples.

Polymorphism Analysis of MUC2- MS7 in Normal Subjects repeats size (bp) N = 1088 Frequency repeats size (bp) N = 1088 Frequency 29 2159 4 0.004 88 6230 2 0.002 38 2780 2 0.002 91 7437 8 0.008 40 2918 16 0.015 97 6851 5 0.005 42 3056 206 0.188 100 7058 2 0.002 44 3194 One 0.001 101 7127 One 0.001 50 3608 4 0.004 102 7196 16 0.015 57 4091 2 0.002 103 7265 2 0.002 61 4367 One 0.001 104 7334 792 0.792 70 4988 5 0.005 105 7403 7 0.007 74 5264 2 0.002 108 7610 2 0.002 78 5540 One 0.001 113 7955 2 0.002 84 5954 2 0.002 118 8300 One 0.001 128 8990 2 0.002

As a result of examining 544 normal people, various alleles of various sizes ranging from 2159 bp to 8990 bp were identified, and the repeated sequence of 69 bp was repeated variously from 29 to 128 times (Table 3 and FIG. 3).

Example 2: Determining the Genetic Delivery of Polymorphic Sociability (minisatellite, MS) through meiosis

Genomic DNA was extracted from blood of the grandmother, parent, and offspring, and PCR was performed in the same manner as in Example 1 to confirm the allele of MUC2- MS7.

As a result, it was confirmed that the MUC2- MS7 region is genetically correctly transmitted through meiosis between parents and offspring (FIG. 5). The first and last lanes are size markers. 1 or 2 means grandfather or grandmother, 3, 4 or 7, 8 means father or mother, and DNA samples of progeny are shown as 5, 6 and 9. This indicates that the so-called within the MUC2 gene is inherited by Mendel's law and can be used as an efficient marker for paternity or forensic sentiment, and whether the same disease occurs in a family using DNA typing markers. That means you can predict.

Example 3 Measurement of Association with Gastric Cancer in the MUC2- MS7 Region

Reported that the structural characteristics of Tandem repeats (TR) are involved in gene expression, mainly on h-Ras , to investigate the effect of TR region on the expression of MUC2 genes in normal and gastric cancer patients. Genomic DNA was extracted and subjected to PCR in the same manner as in Example 1 to compare haplotype patterns between normal and gastric cancer patients.

Alleles appearing in less than 1% of normal populations were called rare alleles. The rare alleles of MUC2- MS7 and susceptibility to cancer development were analyzed (Table 4).

Polymorphism Analysis of MUC2- MS7 in Gastric Cancer Patients. repeats size (bp) N = 720 Frequency repeats size (bp) N = 720 Frequency 40 2159 10 0.014 91 6437 2 0.003 42 2780 102 0.140 97 6851 2 0.003 47 3056 2 0.003 102 7196 10 0.014 50 3194 3 0.004 103 7265 5 0.007 57 3608 13 0.018 104 7334 541 0.751 70 4367 8 0.011 105 7403 3 0.004 74 4988 3 0.004 108 7610 3 0.004 84 5540 One 0.001 113 7955 12 0.017 118 8300 One 0.001

In the case of MUC2- MS7, alleles were divided into patterns, the patterns consisting only of common alleles were designated as C / C, and the patterns having at least one rare allele were divided by R /-.

As a result, it was confirmed that in patients with gastric cancer, the R /-pattern was about 1.6 times higher than normal. This means that with a rare allele of MUC2- MS7, sensitivity to gastric cancer is about 1.6 times higher than normal. As a result of statistical analysis, the relative risk of gastric cancer was found to be 1.02 ~ 2.46 ( p = 0.04) at 95% confidence interval when there was at least one rare allele of MUC2- MS7 (Table 5, FIG. 4). . This means that the rare allele of MUC2- MS7 is associated with gastric cancer.

MUC2- MS7 Rare Allele Pattern Between Normal and Gastric Cancer Patients MUC2 -MS7 Total case C / C C / R + R / R OR (95% CI) p Control (%) 544 500 (91.9) 44 (30 + 14) (8.1) 1.00 (Reference) Gastric cancer (%) 360 316 (87.8) 46 (40 + 14) (12.2) 1.58 (1.02-2.46) 0.04

 C: common allele, R: rare allele

The rare alleles were divided by length to compare 29-78 repetitions with short rare alleles and 84-128 repetitions with long rare alleles.

As a result, as shown in Table 6, in the case of MUC2- MS7 gastric cancer patients have a short rare allele, it was found that the risk of gastric cancer is 2.09 times higher than that of normal susceptibility.

Through the above results, MUC2- MS7 is an important marker to investigate the sensitivity to gastric cancer, it can be seen that it is used as an important data for predictive diagnosis.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

1 is a schematic diagram showing the position and direction of the mucin gene located at the terminal of human chromosome 11 (A) is the result of analyzing the band structure of chromosome 11 by MAP Viewer of NCBI website, (B) is a diagram of the mucin gene on the 11p15.5 chromosome, MUC2 , MUC5AC and MUC5B is The MUC6 is towards the telomere, while towards the centromere.

Figure 2 is a schematic showing the structure of the MUC2 gene and the location of minisatellite in the gene, exon (exon) is shown in blue lines, the location of the so-called (minisatellite) detected by the Tandem Repeats Finder Program with an asterisk (*) Indicated.

Figure 3 is an electrophoretic picture showing the MUC2- MS7 polymorphism in the normal group.

Figure 4 is an electrophoresis picture showing the MUC2- MS7 polymorphism in the gastric cancer patient group.

Figure 5 is an electrophoretic picture showing MUC2- MS7 is genetically transmitted between parent and offspring, the first and last lanes are size markers, 1 or 2 is grandfather or grandmother, 3,4 or 7,8 is father Or mother, and DNA samples of progeny are shown as 5, 6 and 9, respectively.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Minisatellites of MUC2 Gene and DNA Typing Kits and Diagnostic          Kits for Cancer Using the Same <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> minisatellite <400> 1 ccaaccacga cacccatcac caccaccact acggtgaccc caaccccaac acccaccggc 60 acacagacc 69 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 catcaccacc accactacgg tgac 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cggaggattg gatgtggtca actc 24  

Claims (9)

delete delete delete delete (A) A primer for detecting polymorphic so-called MUC2-MS7 of SEQ ID NO: 2 and SEQ ID NO: 2 and SEQ ID NO: 3 after DNA denaturation using genomic DNA isolated from tissues of an individual, for 45 seconds at 94 ° C and 15 seconds at 65 ° C. Performing PCR with 28 cycles for 5 minutes at 72 ° C .; (B) separating the PCR product by electrophoresis to identify the polymorphic so-called MUC2-MS7; And (C) DNA typing the polymorphic so-called MUC2-MS7. 6. The method of claim 5, wherein said DNA typing is used to identify the likelihood of developing the same cancer in a family. delete 7. The method of claim 5 or 6, wherein the cancer is gastric cancer. delete

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