KR101100437B1 - A polynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucleotide - Google Patents

Abstract

The present invention provides a polynucleotide comprising at least 10 contiguous nucleotides derived from the group consisting of SEQ ID NOs: 1 to 12, and comprising a 101 st base or a complementary polynucleotide thereof.

Description

A polynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucleotide}

The present invention relates to a polynucleotide for use related to colon cancer, a microarray comprising the same, a diagnostic kit, and a method for diagnosing colon cancer.

The genomes of all organisms undergo spontaneous mutation that causes a mutant sequence of the offspring in the course of evolution (Gusella, Ann. Rev. Biochem . 55, 831-854 (1986)). Variants can be evolutionarily beneficial or disadvantageous or neutral compared to their ancestral form. In some cases, the variant is not passed on to the offspring due to a fatal penalty. In other cases, it gives the species an evolutionary benefit, and eventually DNA is inserted into most of the species, effectively ancestral forms. In many cases, these ancestral morphologies and variants survive and coexist among the species. Polymorphism occurs due to the coexistence of plural forms of sequences.

Examples of such polymorphisms include restriction fragment length polymorphism (RFLP), short tandem repeats (STR), variable number tandem repeats (VNTR), and single nucleotide polymorphism (SNP). Double SNPs are monobasic polymorphs and take the form of single nucleotide variations between individuals of the same species. When monobasic polymorphism occurs in the coding region sequence, a defective or mutated protein may be expressed by any of the polymorphic forms. In other cases, a single base polymorphism may occur in the non-coding region sequence. Some of these can result in the expression of defective or mutated proteins (eg, as a result of defective splicing). Some monobasic polymorphisms may have no effect on the phenotype.

Monobasic polymorphism is known to occur at a frequency of about once every 1,000 bp in humans. When these monobasic polymorphisms affect a phenotype such as a disease, the polynucleotide comprising the monobasic polymorphism can be used as a primer or probe in diagnosing such diseases. Monoclonal antibodies that specifically bind to the monobasic polymorph can also be used for diagnosis of diseases. Currently, several research institutes are conducting research on single base analysis and its function analysis. The single-base polymorphic nucleotide sequence and other experimental results found in this way are made into a database and published, so that anyone can access and use it for research.

However, these single-base polymorphisms simply found that single-base polymorphisms exist on the human genome or cDNA, but they did not reveal their effect on the phenotype. Some of these are known for their function, but most of them are unknown.

Colorectal cancer is a very common cancer in the world including our country. In Korea, it is the fourth most common cancer in both men and women, and the mortality rate from cancer is the fourth, and about 7 people per 100,000 people die of colon cancer. The mortality rate has also increased by about 80% in the last 10 years.

It is known that the proportion of environmental factors is large in the occurrence of colon cancer, and the main causes include rapid westernization of diet and excessive consumption of animal fat or protein. However, it is known that around 5% of colon cancer is caused by a genetic predisposition.

More than 90% of the patients are 40 years of age or older, and it is more common in cases of family history of colon cancer, inflammatory bowel disease, colon polyps, ovarian cancer, uterine cancer, and breast cancer with age (high-risk group). Is known. When it occurs in young people in their 30s and 40s, it is often caused by a genetic predisposition.

Colorectal cancer has a cure rate close to 100% when detected early, but it is difficult to detect early because there are generally no characteristic subjective symptoms. In order to detect colon cancer in the asymptomatic period, a screening test, an occult blood test (a test that can confirm even a small amount of bleeding in the stool), is used to select a person who needs a close examination. However, this method is not suitable for use in early diagnosis because of its high false-positive and false-negative rates. Currently, methods such as colonoscopy, colonoscopy, and radiation diagnosis are used to confirm the diagnosis of colon cancer. A tumor marker called CEA is commonly used, but it is only used as an indicator for determining the progression of colorectal cancer and the therapeutic effect, and a proven tumor marker that can detect or predict colorectal cancer early through blood tests is still available. It is not known to exist. There are several known markers for selecting patients in a high-risk group who are susceptible to colorectal cancer or for early diagnosis, but their use has a disadvantage that is not suitable for selecting the majority of colorectal cancer patients.

The biggest problem in diagnosing or early predicting various cancers and complex diseases, including conventional colon cancer, is that these diseases must progress to some extent before they can be diagnosed using a physical method. However, as recent advances in various molecular biology technologies and studies on human genomes are primarily completed, genes or genetic mutations directly or indirectly related to diseases can be found and used for early diagnosis of diseases. Early diagnosis has become possible to predict the onset of disease with genetic factors from existing diagnostic methods that depended on phenotypic or external characteristics of the disease. There are biochemical and molecular biological methods for diagnosing colorectal cancer that have been used in the past. Among them, in the case of colorectal cancer diagnosis by molecular biological methods, early diagnosis at the level desired by patients or specialists is made due to the lack of information on the gene or genetic mutation related to the disease and the relationship with the incidence of colorectal cancer. I can't. In addition, in most cases where one biological marker is used for diagnosis, it is rare that both the sensitivity and specificity of the marker have high values, and in the case of high sensitivity, the specificity is low or vice versa. to be. Therefore, since there is a high possibility that errors may occur during diagnosis, there are many cases in which a specialist cannot secure a desired level of accuracy, and most are used as a diagnostic marker of a pre-screening concept for precise diagnosis.

It is an object of the present invention to provide a polynucleotide containing a monobasic polymorph associated with colorectal cancer.

Another object of the present invention is to provide a microarray or a kit for diagnosing colorectal cancer comprising a polynucleotide containing a single nucleotide polymorphism related to colorectal cancer.

Another object of the present invention is to provide a method for diagnosing colorectal cancer using the polynucleotide associated with colorectal cancer of the present invention.

The present invention is a polynucleotide comprising 10 or more contiguous nucleotides derived from the group consisting of the polynucleotides of SEQ ID NOs: 1 to 12, and a polynucleotide comprising a polymorphic site (n) base at position 101 or a complementary polynucleotide thereof Provides.

The length of the polynucleotide includes 10 or more nucleotides including the polymorphic sites of SEQ ID NOs: 1 to 12 (positions 101: each indicated by "n". The same hereinafter). The length is 10 to 200 nucleotides, preferably 10 to 100 nucleotides, more preferably 10 to 50 nucleotides.

The polynucleotide having the nucleotide sequence of SEQ ID NO: 1 to 12 is a polymorphic sequence. The polymorphic sequence refers to a sequence including a polymorphic site representing monobasic polymorphism in a nucleotide sequence. Polymorphic site refers to a site in which monobasic polymorphism occurs among polymorphic sequences. The polynucleotide can be DNA or RNA.

In the present invention, the polymorphic site (n) of the polymorphic sequence at position 101 of SEQ ID NO: 1 to 12 is associated with colon cancer. This could be confirmed through the results of sequencing DNA obtained from blood samples derived from colon cancer patients and normal people. The results are shown in Tables 1, 2 and 3 below.

Table 1 and 2. Association between the polymorphic sequence of SEQ ID NO: 1 to 12 and colon cancer

ASSAY_ID SNP Sequence number Allele frequency Number of genotype appearances cas_A2 con_A2 Delta cas_
A1A1 cas_
A1A2 cas_
A2A2 con_
A1A1 con_
A1A2 con_
A2A2 CCK048 [A/C] One 0.945 0.973 0.028 0 25 204 0 16 277 CCK061 [A/G] 2 0.646 0.714 0.068 31 101 98 22 120 145 CCK117 [A/C] 3 0.636 0.555 0.081 24 107 82 51 157 83 CCK162 [G/C] 4 0.647 0.714 0.067 31 99 98 21 120 142 CCY_041 [T/G] 5 0.61 0.507 0.103 32 109 81 67 140 71 CCY_056 [A/T] 6 0.39 0.299 0.091 106 60 57 144 120 27 CCY_065 [T/G] 7 0.409 0.328 0.081 84 105 42 132 126 32 CCY_067 [A/C] 8 0.377 0.286 0.091 97 85 42 147 123 22 CCY_071 [G/T] 9 0.754 0.821 0.067 34 45 151 13 77 197 CCY_093 [G/T] 10 0.413 0.478 0.065 74 121 34 81 140 68 CCY_202 [G/A] 11 0.355 0.285 0.07 103 106 33 148 123 22 CCY_205 [A/G] 12 0.631 0.704 0.073 33 108 95 21 123 135

Association; chi-square (df=2) Odds ratio (OR): multiple models HWE status call rate Chi_
value Chi_exact
_p-Value Risk antagonist OR CI cas_HW cas_HW cas_call_rate con_call_rate 5.287
6.041
7.299
6.155
10.754
27.984
7.34
14.733
17.789
6.166
6.729
7.056 3.20E-02
4.88E-02
2.60E-02
4.61E-02
4.62E-03
8.38E-07
2.55E-02
6.32E-04
1.37E-04
4.58E-02
3.46E-02
2.94E-02 A1 A
A1 A
A2 C
A1 G
A2 G
A2 T
A2 G
A2 C
A1 G
A1 G
A2 A
A1 A 2.06
1.37
1.41
1.36
1.52
1.49
1.43
1.52
1.49
1.30
1.39
1.39 (1.085,3.9)
(1.055,1.785)
(1.085,1.812)
(1.044,1.774)
(1.182,1.961)
(1.156,1.946)
(1.103,1.832)
(1.164,1.965)
(1.102,2.012)
(1.016,1.666)
(1.068,1.792)
(1.071,1.805)) .174, HWE
.569, HWE
1.584, HWE
.657, HWE
.195, HWE
44.441, HWD
.945, HWE
9.12, HWD
56.139, HWD
1.747, HWE
.535, HWE
.083, HWE .067, HWE
.127, HWE
2.407, HWE
.419, HWE
.029, HWE
.075, HWE
.071, HWE
.185, HWE
2.444, HWE
.287, HWE
.185, HWE
.863, HWE 0.99
One
0.92
0.99
0.87
0.87
0.9
0.88
0.9
0.89
0.95
0.92 One
0.98
0.99
0.97
0.94
0.98
0.98
0.99
0.97
0.98
0.99
0.94

Table 3. Characteristics of the polymorphic sequence of SEQ ID NO: 1-12

ASSAY_ID rs chromosome location band gene Explanation SNP
role amino acid
change CCK048 rs2863383 3 167396140 3q26.1 gene
between - gene
between No change CCK061 rs7151139 14 21933597 14q11.2 C14orf120 Chromosome 14 orf 120 Intron No change CCK117 rs724454 4 91421840 4q22.1 gene
between - gene
between No change CCK162 rs10142383 14 21932663 14q11.2 C14orf120 Chromosome 14 orf 120 Intron No change CCY_041 rs1402026 5 114159705 5q22.3 gene
between - gene
between No change CCY_056 rs1485217 3 3917830 3p26.2 gene
between - gene
between No change CCY_065 rs1996489 3 167399578 3q26.1 gene
between - gene
between No change CCY_067 rs2236261 14 21934642 14q11.2 C14orf120 Chromosome 14 orf 120 Coding-sinon,reference No change CCY_071 rs1340655 10 61325850 10q21.2 ANK3 Anclean 3, Ranbier node
(Anclean G) Intron No change CCY_093 rs1334856 13 82206386 13q31.1 gene
between - gene
between No change CCY_202 rs6573195 14 21934148 14q11.2 C14orf120 Chromosome 14 orf 120 Intron No change CCY_205 rs2295706 14 21935494 14q11.2 C14orf120 Chromosome 14 orf 120 Intron No change

In Tables 1,2 and 3, the meaning of each column is as follows.

ㆍAssay_ID represents the name of the marker.

ㆍSNP refers to the base observed in the single-base polymorphic site, where A1 and A2 are of low mass in the process of sequencing by the homogeneous MassEXTEND (homogeneous MassEXTEND: hereinafter referred to as hME) technique of Sequenom. The allele is named A1 and the allele with a large mass is A2 for convenience of experimentation.

-The sequence number shows the sequence number of the sequence containing each SNP site, and shows the sequence containing the A1 and A2 alleles at the 101st position, respectively.

ㆍIn the allele frequency column, cas_A2, con_A2 and Delta are the frequencies of the A2 alleles in the case sample, the frequency of the A2 alleles in the normal sample, and the difference between cas_A2 and con_A2, respectively. It represents the absolute value of. Where cas_A2 is (frequency of A2A2 genotype x2 + frequency of A1A2 genotype)/(number of samples in disease group x2) in disease group, and con_A2 is (frequency of A2A2 genotype x2 + frequency of A1A2 genotype)/(sample from normal group) in the disease group Number x2).

ㆍGenotype frequency represents the frequency of each genotype, and cas_A1A1, cas_A1A2 and cas_A2A2 and con_A1A1, con_A1A2 and con_A2A2 represent the number of people with A1A1, A1A2 and A2A2 genotypes in the disease group and the normal group, respectively.

ㆍChi-square (df=2) represents a chi-square value with a degree of freedom of 2, and Chi-value is a chi-square result and is a standard value for p-value calculation. The chi-exact-p-value represents the p-value of Fisher's exact test of chi-square test. It is a variable used to test more accurate statistical significance (p-value) through test. In the present invention, when the p-value ≤ 0.05, the genotypes of the disease group and the normal group are not the same, that is, it is determined that it is significant.

ㆍAs for the risk allele, if the reference allele is A2, and the frequency of A2 in the disease group is greater than the frequency of A2 in the normal group (ie cas_A2> con_A2), A2 is the risk allele, and the In the opposite case, A1 was used as the risk allele.

ㆍOdds ratio represents the ratio of the probability of having a risk allele among the disease group to the probability of having a risk allele among the normal group. In the present invention, the Mantel-Haenszel odds ratio method was used. CI represents the 95% confidence interval of the odds ratio and represents (lower confidence interval, upper confidence interval). If the confidence interval contains 1, the association with disease cannot be judged to be significant.

ㆍHWE state represents the state of Hardy-Weinberg Equilibrium, and con_HWE and cas_HWE represent the state of Hardy-Weinberg equilibrium in the normal group and the disease group. In the chi-square (df=1) test, based on chi-value = 6.63 (p-value =0.01, df=1), and if it is greater than 6.63, it is Hardy-Weinberg Disequilibrium (HWD), which is less than 6.63. In small cases, it was judged as Hardy-Weinberg Equilibrium (HWE).

ㆍCall rate (call_rate) represents the number of samples in which genotype was successfully tested with respect to the total number of samples put into the original experiment, and cas_call_rate and con_call_rate are among the total samples (300 people) used in the experiment in the disease group and the normal group, respectively. It represents the percentage at which the genotype was successfully analyzed.

ㆍ The rs number means the NCBI dbSNP identification number.

Tables 1, 2 and 3 describe the characteristics associated with each SNP marker based on NCBI build 119 on February 1, 2005.

As shown in Tables 1, 2, and 3, the polymorphic markers of SEQ ID NOs: 1 to 12 of the present invention are Chi-exact-p-value values at 95% reliability as a result of χ square analysis between the expected and observed values of the frequency of occurrence of alleles. The range of 0.0000008 to 0.049 was significantly different from that of the control group, and the range of odds ratio was 1.30 to 2.06, indicating that it was associated with colon cancer.

Therefore, the polynucleotide of the present invention can be effectively used for diagnosis, gene fingerprint analysis or treatment related to colon cancer. Specifically, it can be used as a primer or probe for diagnosis of colon cancer. In addition, it can be used as an anti-sense DNA or composition for the treatment of colorectal cancer.

The present invention also provides a polynucleotide comprising 10 or more contiguous nucleotides derived from SEQ ID NOs: 1 to 12 including a polymorphic site, or an allele-specific polynucleotide for diagnosis of colorectal cancer that hybridizes with a complement thereof.

The allele-specific polynucleotide means specifically hybridizing to each allele. That is, it refers to hybridization so that the base of the polymorphic site in each polymorphic sequence of SEQ ID NOs: 1 to 12 can be specifically distinguished. Here, hybridization is usually carried out under stringent conditions, for example, a salt concentration of 1M or less and a temperature of 25°C or more. For example, 5xSSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and conditions of 25 to 30° C. may be suitable for allele-specific probe hybridization.

In the present invention, the allele-specific polynucleotide may be a primer. Herein, "primer" refers to a template-directed DNA synthesis under appropriate conditions (eg, 4 different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer. It refers to a single-stranded oligonucleotide that can serve as a starting point. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the template, but must be sufficiently complementary to hybridize to the template. It is preferable that the 3'end of the primer is aligned with the polymorphic site (base n) of SEQ ID NOs: 1 to 12. The primer hybridizes to a target DNA containing a polymorphic site, and the primer initiates amplification of an allelic form showing complete homology. This primer is used in pairs with a second primer that hybridizes to the opposite side. The product is amplified from the two primers by amplification, meaning that a specific allelic form is present. The primer of the present invention includes a polynucleotide fragment used in a ligase chain reaction (LCR).

In the present invention, the allele-specific polynucleotide may be a probe. In the present invention, "probe" means a hybridization probe, and means an oligonucleotide capable of sequence-specific binding to a complementary strand of a nucleic acid. Such probes include the peptide nucleic acids described in Nielsen et al., Science 254, 1497-1500 (1991). The probe of the present invention is an allele-specific probe, and a polymorphic site exists in a nucleic acid fragment derived from two members of the same species, and hybridizes to a DNA fragment derived from one member, but does not hybridize to a fragment derived from another member. . In this case, the hybridization conditions show a significant difference in hybridization strength between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. In the probe of the present invention, it is preferable that the central portion (ie, position 7 if it is a 15 nucleotide probe, position 8 or 9 if it is a 16 nucleotide probe) is aligned with the polymorphic region of the sequence. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used in a diagnostic method or the like for detecting alleles. The diagnostic methods include detection methods based on hybridization of nucleic acids such as Southern blotting, and may be provided in a form that is previously bound to a substrate of a DNA chip in a method using a DNA chip.

The present invention also includes a microarray for colon cancer diagnosis comprising the polynucleotide of the present invention or a complementary polynucleotide thereof. The microarray may include DNA or RNA polynucleotides. The microarray is made of a conventional microarray, except for including the polynucleotide of the present invention.

The present invention also provides a kit for diagnosing colon cancer comprising the polynucleotide of the present invention. The kit may contain not only the polynucleotide of the present invention, but also a reagent necessary for a polymerization reaction, such as dNTP, various kinds of polymerases, and coloring agents.

In addition, the present invention comprises the steps of obtaining a nucleic acid sample from a subject; And

It provides a method for diagnosing colorectal cancer comprising determining the nucleotide sequence of the polymorphic site (n) at one or more of the 101 positions of the polynucleotides of SEQ ID NOs: 1 to 12 or complementary polynucleotides thereof. Here, as shown in Tables 1, 2, and 3, when the nucleotide sequence of the polymorphic site is a risk allele, determining that the subject is a colorectal cancer patient or has a high probability of developing colorectal cancer.

Among the above methods, the step of first obtaining a nucleic acid from a subject may be performed by a conventional DNA separation method. For example, it can be obtained by amplifying a target nucleic acid through PCR and purifying it. Other Ligase Chain Reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc . Natl . Acad . Sci . USA) 86, 1173 (1989)) and self-maintained sequence replication (Guatelli et al., Proc . Natl . Acad . Sci . USA 87, 1874 (1990)) and nucleic acid-based sequence amplification (NASBA) can be used. The last two methods are related to isothermal reactions based on isothermal transcription, and produce 30 or 100-fold single-stranded RNA and double-stranded DNA as amplification products.

* In one embodiment of the method of the present invention, the step of determining the nucleotide sequence of the polymorphic site is a polynucleotide comprising 10 or more contiguous nucleotides derived from the group consisting of SEQ ID NOs: 1 to 12 of the present invention, the 101 th Hybridizing the nucleic acid sample to a microarray on which a polynucleotide for diagnosis or treatment of colorectal cancer containing a base or a complementary polynucleotide thereof is immobilized; And detecting the hybridization result.

Methods of preparing microarrays by immobilizing microarrays and probe polynucleotides on a substrate are well known in the art. The process of immobilizing the probe polynucleotide associated with colorectal cancer of the present invention onto a substrate can also be easily prepared using such a conventional technique. In addition, the detection of hybridization and hybridization results of nucleic acids on microarrays is well known in the art. The detection is performed by using a nucleic acid sample with a detectable signal including a fluorescent substance such as Cy3 and Cy5. The hybridization result can be detected by labeling with a labeling substance capable of generating, then hybridizing on a microarray and detecting a signal generated from the labeling substance.

According to the polynucleotide of the present invention, it can be used for purposes related to colorectal cancer, such as diagnosis, treatment, and gene fingerprint analysis of colorectal cancer.

Further, according to the microarray and kit comprising the polynucleotide of the present invention, colon cancer can be effectively diagnosed.

According to the method for analyzing polynucleotides related to colon cancer of the present invention, the presence or absence of a risk of colon cancer can be effectively diagnosed.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

Example 1

In this example, DNA was isolated from the blood of a group of patients (300 people) who were found to be patients with colorectal cancer among Koreans, and who were in the same age group as the patient group and who had not yet had symptoms of colorectal cancer. The frequency of occurrence of nucleotide polymorphism was analyzed. The single base polymorphism selected in this example is a public database (NCBI dbSNP: http://www.ncbi.nlm.nih.gov/SNP/ ) or Sequenom's realsnp.com (http:://www.realsnp.com) /), and the single base sequence in the sample was analyzed using primers using sequences around the selected single base polymorphism.

1. Preparation of DNA sample

DNA was extracted from blood collected from colon cancer patients and normal people. DNA extraction was performed according to a known extraction method (Molecular cloning: A Laboratory Manual, p 392, Sambrook, Fritsch and Maniatis, 2nd edition, Cold Spring Harbor Press, 1989) and instructions of a commercial kit (Gentra system). Among the extracted DNA, only those with a UV measurement value (260/280 nm) of at least 1.6 were selected and used.

*2. Amplification of target DNA

A target DNA, which is a constant DNA region containing a single base polymorph to be analyzed, was amplified using PCR. The PCR method was carried out by a conventional method, and the conditions were as follows. First, the target genomic DNA was prepared at 2.5 ng/ml. Next, the following PCR reaction solution was prepared.

Water (HPLC grade) 2.24µl

10x buffer (containing 15 mM MgCl ₂ , 25 mM MgCl ₂ ) 0.5 μl

dNTP mix (GIBCO) (25 mM/each) 0.04 μl

Taq pol (HotStar) (5U/µl) 0.02µl

Pre/post primer mix (1 μM/each) 0.02 μl

DNA 1.00µl

Total reaction volume 5.00 μl

Here, the forward and reverse primers were selected at appropriate positions from upstream and downstream of the monobasic polymorph of a known data base. The primers are summarized in Table 4.

The thermal cycling reaction was maintained at 95° C. for 15 minutes, repeated at 95° C. for 30 seconds, 56° C. 30 seconds, and 1 minute at 72° C. 45 times, maintained at 72° C. for 3 minutes, and then at 4° C. Kept. As a result, a target DNA fragment having a length of 200 nucleotides or less was obtained.

3. Analysis of single base polymorphism in amplified target DNA

The analysis of the single base polymorphism in the target DNA fragment was performed using the homogeneous MassEXTEND (homogeneous MassEXTEND: hereinafter referred to as hME) technique of Sequenom. The principle of the MassEXTEND technique is as follows. First, a primer (also called an extension primer) that is complementary to the base just before the single base polymorphism in the target DNA fragment is prepared. Next, the primer is hybridized to the target DNA fragment and a DNA polymerization reaction is caused. In this case, a reagent (e.g., ddTTP) is included in the reaction solution to stop the reaction after a base complementary to the first allele base (e.g., A allele) is added to the target monobasic polymorphic allele. Let it. As a result, when the first allele (e.g., A allele) is present in the target DNA fragment, only one base (e.g., T) complementary to the first allele is added. . On the other hand, when a second allele (e.g., G allele) is present in the target DNA fragment, the closest first allele to which a base (e.g., C) complementary to the second allele is added. You get a product that extends to the base (eg, A). By determining the length of the product extended from the primer thus obtained through mass spectrometry, the kind of alleles present in the target DNA can be determined. The specific experimental conditions were as follows.

First, dNTPs that do not bind were removed from the PCR product. To this end, 1.53 µl of pure water, 0.17 µl of HME buffer, and 0.30 µl of SAP (shrimp alkaline phosphatase) were added to a 1.5 ml tube and mixed to prepare an SAP enzyme solution. The tube was centrifuged for 10 seconds at 5,000 rpm. Thereafter, the PCR product was put into the SAP solution tube, sealed, and maintained at 37°C for 20 minutes and 85°C for 5 minutes, and then stored at 4°C.

Next, a homogeneous extension reaction was performed using the target DNA product as a template. The reaction solution was as follows.

1.728 µl of water (nano-grade pure water)

0.200 μl of hME extension mix (10x buffer containing 2.25 mM d/ddNTPs)

Extension primer (100 μM each) 0.054 μl

Thermosequenase (32U/µl) 0.018µl

Total volume 2.00µl

After mixing the reaction solution well, it was spin-down centrifuged. After sealing the tube or plate containing the reaction solution well, holding it at 94°C for 2 minutes, repeating 5 seconds at 94°C, 5 seconds at 52°C, and 5 seconds at 72°C 40 times, and then 4°C. Stored in. The homogeneous extension reaction product thus obtained was washed with resin (SpectroCLEAN). The extension primers used in the extension reaction are shown in Table 4.

Table 4. Primers used for target DNA amplification and extension primers used for homogeneous extension reactions

Marker name Target DNA amplification primer (SEQ ID NO) Extension primer
(SEQ ID NO:) Forward primer Reverse primer CCK048 13 14 15 CCK061 16 17 18 CCK117 19 20 21 CCK162 22 23 24 CCY_041 25 26 27 CCY_056 28 29 30 CCY_065 31 32 33 CCY_067 34 35 36 CCY_071 37 38 39 CCY_093 40 41 42 CCY_202 43 44 45 CCY_205 46 47 48

The obtained extension reaction product was sequenced at the polymorphic site using MALDI-TOF (Matrix Assisted Laser Desorption and Ionization-Time of Flight) in the mass spectrometry method. The MALDI-TOF is based on the principle of analyzing the mass by calculating the time taken to fly from the vacuum state to the detector on the other side by flying with the ionized matrix (3-Hydorxypicolinic acid) when the material to be analyzed receives a laser beam. Works. Substances with a small mass reach the detector quickly. Based on the difference in mass and the known monobasic polymorphic sequence, the single nucleotide polymorphic sequence of the target DNA can be determined.

The results of determining the sequence of the polymorphic site of the target DNA using MALDI-TOF as described above are shown in Tables 1, 2, and 3. At this time, each allele may exist in the form of a homozygote or a heterozygote in each individual. According to Mendel's law of genetics and Hardy-Weinberg's law, the composition of alleles constituting a group is maintained at a constant frequency, and if statistically significant, it can give meaning to biological function. As shown in Tables 1, 2 and 3, the single-base polymorphism of the present invention appeared at a statistically significant level in colorectal cancer patients, and it was found that it can be used for diagnosis of colorectal cancer.

The sequences referred to herein are attached in the form of a sequence listing file. The sequence listing file should be construed as part of this specification.

Attach electronic file of sequence list

Claims (6)

A polynucleotide comprising at least 10 contiguous nucleotides derived from SEQ ID NO: 8, comprising a polynucleotide comprising a 101st base or a complementary polynucleotide thereof. A polynucleotide comprising at least 10 consecutive nucleotides derived from SEQ ID NO: 8, comprising a polynucleotide comprising a 101st base or a complementary polynucleotide thereof. Providing a nucleic acid sample isolated from the subject; And
A method for obtaining data for diagnosing colorectal cancer, comprising determining the nucleotide sequence of the polymorphic site (101st) of at least one polynucleotide of SEQ ID NO: 8 or a complementary polynucleotide thereof. 4. The method of claim 3, wherein determining the nucleotide sequence of the polymorphic site comprises hybridizing the nucleic acid sample to the microarray according to claim 1; And
Detecting the hybridization result. 4. The method of claim 3, comprising determining the nucleotide sequence of the polymorphic site and determining if the nucleotide of the polymorphic site of SEQ ID NO: 8 is a risk allele of C. The method of claim 3, comprising determining the nucleotide sequence of the polymorphic site and determining whether the polymorphic site of SEQ ID NO: 8 is CC or CA, or AA.