Mlh1 gene promoter methylation detection kit

Abstract

In methylation determination of DNA through a bisulfite treatment, there was the problem that cytosine to be detected would be erroneously determined as methylated cytosine when the bisulfite treatment had a poor response.　A method for bisulfite-treating a control nucleic acid containing unmethylated cytosine together with DNA to be detected has been developed. Consequently, it is possible to determine whether cytosine to be detected is methylated, and determine whether the bisulfite treatment is satisfactory.

Description

MLH1 gene promoter methylation detection kit

[0001] The present invention relates to a methylation detection kit for the MLH1 gene promoter, a method for detecting methylation of the MLH1 gene promoter, and a diagnostic aid kit for Lynch syndrome-related tumors.

[0002] Most colorectal cancers are sporadic colorectal cancers that develop later in life due to exposure to environmental factors and the accumulation of gene mutations in the mucosal cells of the colon. For this reason, the incidence of sporadic colorectal cancer increases with age (Non-Patent Literature 1).

[0003] On the other hand, some types of colorectal cancer have a familial accumulation and are collectively referred to as hereditary colorectal cancer. Many of the mutations that cause hereditary colorectal cancer are found in tumor suppressor genes and mismatch repair genes. Generally, hereditary colorectal cancer is characterized by its onset at a young age and a high probability of developing multiple colorectal cancers and multi-organ cancers such as endometrial cancer (Non-patent Literature 1). Therefore, reliably identifying hereditary tumors is important for cancer prevention and early detection in that family.

[0004] Hereditary colorectal cancers include familial adenomatous polyposis (FAP) and Lynch syndrome-related tumors (hereditary non-polyposis colorectal cancer: HNPCC), which are frequently inherited in an autosomal dominant manner (Non-patent Literature 1).

[0005] FAP (Follow-onset Adenoma) is easily diagnosed based on its clinical characteristic of developing more than 100 adenomas in the colonic mucosa. On the other hand, Lynch syndrome-related tumors are thought to be likely to be overlooked in routine clinical practice because they do not show any clinical differences from sporadic colorectal cancer. However, Lynch syndrome-related tumors and sporadic colorectal cancer differ significantly in treatment strategies (surgery, chemotherapy, etc.) and prognostic examination methods (Non-patent Literature 1). Therefore, accurately diagnosing both in the early stages of clinical practice is extremely important.

[0006] Lynch syndrome-related tumors are autosomal dominant inherited disorders primarily caused by mutations in mismatch repair genes, and are usually diagnosed according to the following procedure: First, clinical information is collected from patients suspected of having Lynch syndrome-related tumors, and they are tested to see if they meet Amsterdam Criteria II. If the criteria are met, microsatellite instability (MSI) testing of the tumor tissue is performed (Non-Patent Literature 1). Microsatellite instability refers to the phenomenon in which microsatellites, which are repeat sequences of 1 to several base pairs present in the genome, exhibit a different number of repeats than normal cells due to abnormalities in the mismatch repair mechanism or other reasons.

[0007] Because the Amsterdam Criteria II have strict criteria, false-negative Lynch syndrome-related tumors that do not meet the criteria are often missed. The revised Zevezda guidelines are used to compensate for this drawback. The revised Zevezda guidelines recommend performing the above-mentioned MSI test when 1 to 5 clinical information or findings are met (Non-Patent Literature 2).

[0008] Tumor tissue in Lynch syndrome-related tumors frequently exhibits high-frequency microsatellite instability (high-frequency MSI: MSI-H) (Non-patent Literature 3). Therefore, if the MSI test result in tumor cells is MSI-H, Lynch syndrome-related tumors are strongly suspected.

[0009] However, among patients diagnosed with MSI-H, Lynch syndrome-related tumors account for only about 16%, and sporadic colorectal cancers, which are not Lynch syndrome-related tumors, are found in about 10% to 15% (Non-patent Literature 4). Therefore, unless Lynch syndrome-related tumors and sporadic colorectal cancers are distinguished, MSI testing alone cannot definitively diagnose Lynch syndrome-related tumors.

[0010] One way to distinguish between Lynch syndrome-related tumors and sporadic colorectal cancer is that sporadic colorectal cancer tends to occur more frequently in elderly women and in the right side of the colon. Furthermore, Lynch syndrome-related tumors show deficiencies in the mismatch repair proteins MLH1 and MSH2, while sporadic colorectal cancer almost always shows a deficiency in MLH1 alone. However, neither of these methods can definitively distinguish between the two diseases.

[0011] Non-patent document 5 reported that methylation of CpG islands in the promoter region of the MLH1 gene encoding the MLH1 protein is the cause of MLH1 protein deficiency in sporadic colorectal cancer. On the other hand, methylation in the MLH1 protein promoter is not observed in Lynch syndrome-related tumors. Therefore, it was suggested that by using the blood of a subject and confirming the presence or absence of CpG island methylation in the MLH1 gene promoter as a biomarker, it may be possible to accurately determine whether the subject has sporadic colorectal cancer or Lynch syndrome-related tumors.

[0012] Guidelines for the Treatment of Hereditary Colorectal Cancer, 2024 Edition, Kinbara Publishing Co., Ltd. Umar, A., et al., 2004, Journal of the National Cancer Institute, 96: 261-268. Aaltonen LA, et al., 1994, Cancer Res 54: 1645-1648. Latham A Srinivasan P, Kemel Y, et al.: Microsatellite instability is associated with the presence of Lynch syndrome pan-cancer. J Clin Oncol 37:286-295, 2019. J.G. Herman, et al., 1998, Sci. USA, 95: 6870-6875.

[0013] Generally, the presence or absence of DNA methylation is detected by treating the target cytosine with bisulfite, converting unmethylated cytosine to uracil, and then detecting the difference in bases between the unmethylated and methylated cytosine.

[0014] However, if the bisulfite treatment is unsuccessful, the uracil conversion of unmethylated cytosine does not occur, and it remains as cytosine, leading to the misidentification of the target cytosine as methylated cytosine. This poses a serious problem, as it can lead to misdiagnosis in the definitive diagnosis of sporadic colorectal cancer and Lynch syndrome-related tumors.

[0015] To solve the above problems, the inventors have developed a method of treating a control region containing unmethylated cytosine, along with the detection region, with bisulfite. This method makes it possible to determine whether or not the target cytosine in the detection region is methylated, and to determine the quality of the bisulfite treatment, thereby solving the above problems. The present invention is based on this newly developed method and includes the following.

[0016] (1) A methylation detection kit for the MLH1 gene promoter, comprising: an MLH1 primer set for amplifying a detection region containing a methylation target site of the MLH1 gene promoter; an MLH1 probe that hybridizes to a nucleotide sequence containing the methylation target site in the amplified product of the detection region after bisulfite (BS) treatment and nucleic acid amplification reaction; a control primer set for amplifying a control region containing unmethylated cytosine (n-mC) of a control nucleic acid; a BS-treated detection probe that hybridizes to a nucleotide sequence corresponding to n-mC in the amplified product of the control region after BS treatment and nucleic acid amplification reaction; and a non-bisulfite (n-BS) treated detection probe that hybridizes to a nucleotide sequence corresponding to n-mC in the amplified product of the control region after a nucleic acid amplification reaction without bisulfite treatment, wherein the detection region is all or part of the C region and / or D region of the MLH1 gene promoter. (2) The methylation detection kit according to (1), wherein the MLH1 gene promoter consists of the nucleotide sequence shown in Sequence ID No. 1. (3) The methylation detection kit according to (1), wherein the C region and D region consist of the nucleotide sequences shown in Sequence ID No. 2 and 3, respectively. (4) The methylation detection kit according to any one of (1) to (3), wherein the MLH1 probe consists of an MLH1-mC compatible probe that hybridizes to a nucleotide sequence corresponding to methylated cytosine (mC) at the methylation target site, and / or an MLH1-n-mC compatible probe that hybridizes to a nucleotide sequence corresponding to unmethylated cytosine (n-mC). (5) The methylation detection kit according to any one of (1) to (4), comprising a microarray on which the MLH1 probe, a BS-treated detection probe, and an n-BS-treated detection probe are immobilized. (6) The methylation detection kit according to any one of (1) to (5), wherein the control nucleic acid is the β-actin (ACTB) gene. (7) A methylation detection kit according to any one of (1) to (6), wherein the BS-treated detection probe includes the nucleotide sequence shown in SEQ ID NO: 10, and the n-BS-treated detection probe includes the nucleotide sequence shown in SEQ ID NO: 11. (8) A diagnostic aid kit for Lynch syndrome-related tumors using the methylation detection kit according to any one of (1) to (7).(9) A method for detecting methylation of the MLH1 gene promoter, comprising: a BS treatment step of treating genomic DNA derived from a subject with BS; a control region amplification step of amplifying a control region containing n-mC using a control primer set capable of amplifying the control region, using the n-BS treated genomic DNA before the BS treatment step and the BS treated genomic DNA after the BS treatment step as templates; a control probe binding step of hybridizing a BS treatment detection probe to the BS treated control region amplification product and an n-BS treatment detection probe to the non-BS treated control region amplification product after the control region amplification step; and a control probe measurement step of measuring the amount of each probe bound after the control probe binding step. The method comprises: a detection region amplification step of using the BS-treated genomic DNA as a template to amplify a detection region including predetermined methylation target sites in the C region and / or D region using a primer set capable of amplifying the detection region; an MLH1 probe binding step of hybridizing an MLH1-mC-corresponding probe and an MLH1-n-mC-corresponding probe to the detection region amplification product after the detection region amplification step; an MLH1 probe measurement step of measuring the amount of each probe bound after the MLH1 probe binding step; a BS treatment determination step of determining whether the BS treatment is good or bad based on the binding ratio of the BS-treated detection probe and the n-BS-treated detection probe after the control probe measurement step; and a methylation determination step of determining whether methylation is present or absent at a predetermined methylation target site based on the binding ratio of an MLH1-mC-corresponding probe that hybridizes to the predetermined methylation target site after the MLH1 probe measurement step, when the result of the BS treatment determination step is good. (10) The method of (9) for determining the BS processing determination step, wherein the determination is made using a BS processing determination formula, and if the determination value calculated from the coupling amount of the BS processing detection probe and the coupling amount of the n-BS processing detection probe is higher than a preset cutoff value, the BS processing determination formula is expressed as: BS processing determination value = n-BS processing detection probe coupling amount / (n-BS processing detection probe coupling amount + BS processing detection probe coupling amount).(11) The determination in the methylation determination step is a method in which, using a methylation determination formula, if the determination value calculated from the amount of the MLH1-mC corresponding probe bound and the amount of the MLH1-n-mC corresponding probe bound is higher than a preset cutoff value, it is determined that a predetermined methylation target site is methylated on genomic DNA, wherein the methylation determination formula is expressed as: Methylation determination value = Amount of MLH1-mC corresponding probe bound / (Amount of MLH1-mC corresponding probe bound + Amount of MLH1-n-mC corresponding probe bound) as in (9) or (10). This specification includes the disclosures of Japanese Patent Application No. 2025-008751, which forms the basis of the priority of this application.

[0017] The MLH1 gene promoter methylation detection kit of the present invention allows for easy implementation of the MLH1 gene promoter methylation detection method described later.

[0018] According to the MLH1 gene promoter methylation detection method of the present invention, it is possible to determine whether or not methylation of the MLH1 gene promoter is occurring, and to simultaneously determine the quality of the bisulfite treatment used for methylation detection.

[0019] This figure illustrates the principle and problems of detecting DNA methylation by bisulfite treatment. It schematically shows how to determine the success or failure of bisulfite (BS) treatment using the control probe of the present invention. (a) shows whether each probe binds when the BS reaction is successful, and (b) shows whether each probe binds when the BS reaction is unsuccessful. In the figure, "+" indicates that the probe can bind, and "-" indicates that the probe cannot bind. m"C" indicates methylated cytosine (mC), and the square frame indicates the location of an exemplary control region. This figure schematically shows how to determine the quality of bisulfite (BS) treatment using the test probe of the present invention. (a) shows whether each probe can bind when the detection region contains methylated cytosine at the methylation target site, and (b) shows whether each probe can bind when the detection region does not contain methylated cytosine at the methylation target site. In the figure, "+" indicates that the probe can bind, and "-" indicates that the probe cannot bind. m "C" indicates methylated cytosine (mC), and the square frame indicates the location of an exemplary detection region. This is a flowchart of each step in the MLH1 gene promoter methylation detection method of the present invention. This is a flowchart of the control region performed within the quantitative nucleic acid amplification step (S0403) in the flowchart shown in Figure 4. This is a flowchart of the detection region performed within the quantitative nucleic acid amplification step (S0403) in the flowchart shown in Figure 4. This is a plot diagram showing the relationship between the methylation rate and the judgment value in the MLH1 gene promoter. A shows the result for the promoter C region, and B shows the result for the promoter D region. In the figure, ○ indicates genomic DNA derived from peripheral blood, and ● indicates plasmid DNA used as control nucleic acid. The judgment value (0.1) shown by the dashed line in the figure indicates the cutoff value that separates methylation rates of 0% and 5%.

[0020] 1. MLH1 Gene Promoter Methylation Detection Kit 1-1. Overview A first aspect of the present invention is an MLH1 gene promoter methylation detection kit (often abbreviated as "methylation detection kit" in this specification). The methylation detection kit of the present invention includes an MLH1 primer set, an MLH1 probe, a control primer set, a BS-treated detection probe, and an n-BS-treated detection probe as essential components, and also includes detection reagents and the like as selective components. By using the methylation detection kit of the present invention, it is possible to determine whether or not a specific base is methylated in the MLH1 gene promoter on the subject's genomic DNA, and to simultaneously determine the quality of the bisulfite treatment used for methylation detection.

[0021] 1-2. Definitions of Terms The following terms used herein are defined: The "MLH1 gene" (MutL homolog 1 gene) is the gene that codes for the MLH1 protein, a type of mismatch repair protein. In humans, it is located on chromosome 3 and is the causative gene for Lynch syndrome-related tumors.

[0022] The "MLH1 gene promoter" is the MLH1 gene expression regulatory region located upstream of the coding region of the MLH1 gene on the genomic DNA. In humans, the 1080-base sequence shown in Sequence ID No. 1 corresponds to the wild-type MLH1 gene promoter.

[0023] In this specification, "methylation of the MLH1 gene promoter" refers to the methylation of specific regions (e.g., C region, D region) in the nucleotide sequence of the MLH1 gene promoter. Methylation of the MLH1 gene promoter results in a suppressed expression of the MLH1 gene. Methylation of the MLH1 gene promoter is also found in cancers such as sporadic colorectal cancer (high-frequency microsatellite instability (MSI-H) colorectal cancer), small intestine cancer, endometrial cancer, ovarian cancer, gastric cancer, renal pelvis and ureteral cancer, pancreatic cancer, biliary tract cancer, brain tumors, Muir-Torre syndrome, malignant melanoma, non-small cell lung cancer, prostate cancer, and thyroid cancer.

[0024] In this specification, the "C region" refers to one of four specific regions present in the nucleotide sequence of the MLH1 gene promoter. In the MLH1 gene promoter nucleotide sequence shown in Sequence ID No. 1, this corresponds to the region consisting of the nucleotide sequence shown in Sequence ID No. 2, corresponding to positions 550 to 621. In the human MLH1 gene promoter, there are eight CpG sequences in the C region.

[0025] In this specification, the "D region" refers to one of the specific regions present in the nucleotide sequence of the MLH1 gene promoter, along with the C region. In the nucleotide sequence of the MLH1 gene promoter shown in SEQ ID NO: 1, this region corresponds to the nucleotide sequence shown in SEQ ID NO: 3, which is from position 689 to 813. In the human MLH1 gene promoter, there are seven CpG sequences in the D region.

[0026] In this specification, "methylation" refers to the methylation of DNA. More specifically, it refers to the methylation of cytosine in the CpG sequence on DNA.

[0027] In this specification, "methylated cytosine" (often denoted as "mC" herein) refers to cytosine in a CpG sequence that has been methylated, or the methylated cytosine itself. Specifically, it refers to cytosine in which the carbon at position 5 of the cytosine in the CpG sequence has been methylated by DNA methyltransferase (DNMT) and converted to 5-methylcytosine (5mC).

[0028] In this specification, "non-methylated cytosine" (often denoted as "n-mC") refers to cytosine that is not actually methylated. In principle, this includes cytosines other than those in CpG sequences and cytosines in CpG sequences that are not methylated. However, even cytosines that can be methylated, such as those in CpG sequences, may be referred to as n-mC if, for example, they are actually methylated but not converted to U due to a poor reaction during bisulfite treatment.

[0029] A "CpG sequence" refers to a dinucleotide sequence consisting of a phosphodiester bond between cytosine and guanine. As mentioned above, cytosine in a CpG sequence is the target of methylation.

[0030] A "CpG island" refers to a region on DNA where CpG sequences appear frequently. Assuming there is no bias in the types of bases (A, G, C, and T) appearing in a given genomic region, the probability of a CpG sequence appearing is 1 / 16. Therefore, a CpG island can be defined as a region in a given genomic region where the probability of a CpG sequence appearing exceeds 1 / 16. However, this specification is not limited to this definition, and may also refer to a region in which the GC content exceeds 50% and the proportion of CpG sequences present is 60% or more of the amount expected from the GC content (CpG observed / expected ≥ 0.6).

[0031] Bisulfite treatment (often referred to as "BS treatment" in this specification) is a method of reacting DNA with bisulfite to convert unmethylated cytosine (n-mC) on the DNA to uracil (U). As shown in Figure 1, this base conversion changes the base at that position to thymine (T) after the amplification reaction. On the other hand, methylated cytosine (mC) is not affected by bisulfite and does not undergo base conversion, so its position remains cytosine even after the nucleic acid amplification reaction following the treatment. In this way, by combining the presence or absence of bisulfite treatment with nucleic acid amplification and comparing the respective base sequences, the presence or absence of methylation of cytosine on the DNA can be easily identified.

[0032] In this specification, "detection region" refers to the nucleotide sequence region in the MLH1 gene promoter that is subject to detection for the presence or absence of methylation. Specifically, this refers to the nucleotide sequence region that includes all or part of the C region and / or D region.

[0033] In this specification, "methylation target site" refers to cytosines of CpG sequences that are subject to methylation and are included in the detection region. In particular, this specification includes, but is not limited to, cytosines of CpG sequences that differ in the presence or absence of methylation between Lynch syndrome-related tumors and sporadic colorectal cancers in the detection region.

[0034] As used herein, the "control nucleic acid" refers to a nucleic acid containing a control region for confirming the success or failure of the BS treatment reaction. This nucleic acid is generally DNA.

[0035] As used herein, the "control region" refers to the base sequence region to be detected for confirming the success or failure of the reaction after BS treatment, which is contained in the control nucleic acid. Specifically, a base sequence region containing n-mC, particularly a base sequence region containing one or more cytosines that are not methylated and are not CpG, is applicable.

[0036] As used herein, "simultaneous determination" does not mean determination at the same time, that is, at the same timing, but rather means determining the presence or absence of methylation and also determining the success or failure of the bisulfite treatment used for detecting the methylation.

[0037] 1-3. The methylation detection kit of the present invention includes, as essential components, an MLH1 primer set, an MLH1 probe, a control primer set, a BS treatment detection probe, and an n-BS treatment detection probe. Hereinafter, each component will be specifically described.

[0038] (1) MLH1 primer set A. Basic composition of the MLH1 primer set The "MLH1 primer set" consists of one or more pairs of primers configured to hybridize to the base sequence of a detection region containing a methylation target site in the MLH1 gene promoter and amplify the detection region. The detection region consists of a base sequence region containing all or part of the C region and / or the D region. The cytosine of the CpG sequence, which is the methylation target site, is located at positions 550, 557, 567, 569, 575, 594, 605, and 620 in the C region and at positions 689, 712, 736, 744, 777, 792, and 812 in the D region in the base sequence of the MLH1 gene promoter shown in SEQ ID NO: 1. Therefore, the MLH1 primer set only needs to be able to amplify, as a detection region, a base sequence region containing at least one of these methylation target sites.

[0039] The detection region may be one or more. For example, the detection region may be a single nucleotide sequence containing all or part of the C and D regions, or it may be two nucleotide sequences containing all or part of the C region and all or part of the D region.

[0040] The base length of the region amplified by a pair of MLH1 primer sets is not particularly limited as long as it is long enough to amplify the detection region. The C region is a 72-base sequence shown in SEQ ID NO: 2, the D region is a 125-base sequence shown in SEQ ID NO: 3, and the MLH1 gene promoter is a 1080-base sequence shown in SEQ ID NO: 1. Therefore, for example, if the detection region is a base sequence containing only a portion of the methylation target sites in the C region, it only needs to be 40 bases or longer, and if the entire MLH1 gene promoter is used as the detection region, it only needs to be at least 1080 bases long. Although not limited, it is more preferable that the lengths be 40-1000 bases, 40-800 bases, 50-500 bases, 50-300 bases, 60-270 bases, 60-200 bases, 70-150 bases, or 70-100 bases.

[0041] B. Specific Sequence Examples of MLH1 Primer Sets MLH1 primer sets can be appropriately designed according to common methods in the field, such that the target methylation site is located within the nucleotide sequence region (excluding the primer sequence) that is amplified by the primer set. For example, if the D region of the MLH1 gene promoter sequence is used as the detection region, and the cytosines at positions 736 and 744 in the nucleotide sequence shown in Sequence ID No. 1 are the target methylation sites, the forward (Fw) primer can be designed at a position 5' to the cytosine at position 736, for example, at a distance of 1 nucleotide or more, 3 nucleotides or more, 5 nucleotides or more, 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, or 30 nucleotides or more. Similarly, the reverse (Rv) primer can be designed at a position 3' to the cytosine at position 736 and on the antisense strand (complementary strand), for example, at a distance of 1 nucleotide or more, 3 nucleotides or more, 5 nucleotides or more, 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, or 30 nucleotides or more.

[0042] The Fw primer can be a polynucleotide consisting of 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive base sequences on the sense strand side of the detection region, or a polynucleotide containing one or more deletions, substitutions, or additions of bases in the base sequence of the said polynucleotide, or a polynucleotide containing 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive base sequences on the sense strand side of the detection region. This Fw primer can hybridize to the complementary base sequence on the antisense strand side of the MLH1 gene promoter on genomic DNA.

[0043] The Rv primer can be a polynucleotide consisting of 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive base sequences on the antisense strand side of the detection region, or a polynucleotide containing one or more deletions, substitutions, or additions of bases in the base sequence of the said polynucleotide, or a polynucleotide containing 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive base sequences on the antisense strand side of the detection region. This Rv primer can be hybridized to the complementary base sequence of the MLH1 gene promoter on genomic DNA.

[0044] Each of the aforementioned primers is composed of native nucleotides and / or non-native nucleotides. Typically, they are composed of native nucleotides of DNA or RNA. DNA is particularly preferred because it is highly stable, easy to synthesize, and inexpensive. If necessary, native nucleotides of DNA and RNA can be combined, or non-native nucleotides such as chemically modified nucleic acids or pseudo-nucleic acids can be combined in part. Examples of chemically modified nucleic acids and pseudo-nucleic acids include PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid; registered trademark), methylphosphonate-type DNA, phosphorothioate-type DNA, and 2'-O-methyl-type RNA.

[0045] In the MLH1 primer set, it is preferable that the Fw primer and Rv primer be designed so that their respective Tm values ​​are in the range of 55°C to 80°C or 60°C to 75°C.

[0046] Each primer in the MLH1 primer set may include an additional sequence at its 5' end that differs from the base sequence of the template nucleic acid. The additional sequence can be any base sequence, as long as it does not self-anneal and does not undergo hybridization or other interactions with the target nucleic acid (MLH1 gene promoter in this invention) or the primers or probes included in the methylation detection kit of this invention. Examples include base sequences containing restriction enzyme sites or new primer binding sites.

[0047] As a specific example of an MLH1 primer, if the detection region is the C region and the methylation target sites for detecting the presence or absence of methylation are the cytosines at positions 557, 567, and 569 in the MLH1 gene promoter sequence shown in SEQ ID NO: 1, then for example, a 25-base polynucleotide shown in SEQ ID NO: 12 can be used as the Fw primer for amplifying the MLH1 C region, and a 27-base polynucleotide shown in SEQ ID NO: 13 can be used as the Rv primer.

[0048] Furthermore, if the detection region is the D region, and the methylation target sites for detecting the presence or absence of methylation are the cytosines at positions 736 and 744 in the MLH1 gene promoter sequence shown in SEQ ID NO: 1, then, for example, a 24-base polynucleotide shown in SEQ ID NO: 14 can be used as the Fw primer for MLH1 D region amplification, and a 23-base polynucleotide shown in SEQ ID NO: 15 can be used as the Rv primer.

[0049] C. Labeling of Each Primer in the MLH1 Primer Set Each primer in the MLH1 primer set of the present invention may have some or all of the phosphate groups, sugars, and / or bases of the nucleotides that constitute it labeled with a labeling substance. The labeling position of the labeling substance in the primer may be determined as appropriate according to the characteristics of the labeling substance and the intended use, and is not limited, but the 5' end, which does not contribute to the extension reaction, is usually preferred. Any substance known in the art can be used as the nucleic acid labeling substance. Examples include DIG, fluorescent substances, quenchers, chemiluminescent substances, radioisotopes, biotin, or magnetic beads. The types of labeling in the MLH1 primer and the MLH1 probe are basically the same, and these labels may be used for labeling the primer or probe as needed. Details of each label are described in "C. Labeling of MLH1 Probe" in "(2) MLH1 Probe" below, so the explanation is omitted here.

[0050] (2) MLH1 probe The "MLH1 probe" is a probe that hybridizes to a base sequence containing a methylation target site in the amplified product of the detection region after BS treatment and nucleic acid amplification reaction.

[0051] A. Basic composition of MLH1 probes MLH1 probes are composed of native nucleotides and / or non-native nucleotides. They are usually composed of native nucleotides consisting of DNA only, RNA only, or a hybrid of DNA and RNA, but DNA only is preferred for reasons of chemical stability and ease of chemical synthesis. In addition, all or part of the probe may consist of non-native nucleotides such as PNA (Peptide Nucleic Acid) or BNA (Bridged Nucleic Acid) / LNA (Locked Nucleic Acid; registered trademark).

[0052] The MLH1 probe is not particularly limited, as long as it is designed to specifically hybridize to the nucleotide sequence containing the methylation target site in the amplification product of the detection region. Typically, it is sufficient if it is designed to be a nucleotide sequence complementary to the nucleotide sequence of the detection region. Alternatively, the nucleotide sequence other than the methylation target site may be one that can hybridize to the nucleotide sequence of the detection region under stringent conditions. The Tm value of the MLH1 probe is preferably in the range of 55°C to 80°C, or 60°C to 75°C.

[0053] The base length of the MLH1 probe should be such that it can hybridize to all or part of the base sequence containing cytosine at a specific methylation target site in the amplified product of the detection region after BS treatment and nucleic acid amplification of the MLH1 gene promoter. For example, the probe base length may be one of the following sequences commonly used in this field: 15 to 100 consecutive bases, 15 to 80 consecutive bases, 15 to 60 consecutive bases, 16 to 50 consecutive bases, 17 to 40 consecutive bases, 18 to 30 consecutive bases, or 20 to 25 consecutive bases.

[0054] B. Types of MLH1 Probes and Specific Sequence Examples MLH1 probes consist of MLH1-mC-compatible probes and / or MLH1-n-mC-compatible probes. The "MLH1-mC-compatible probe" hybridizes to the nucleotide sequence corresponding to mC in the detection region of the MLH1 gene promoter. In other words, it is a probe for detecting the MLH1 gene promoter in which a specific cytosine at the methylation target site has been methylated. The "nucleotide sequence corresponding to mC" refers to the nucleotide sequence corresponding to the mC in the amplification product after BS treatment of the MLH1 gene promoter and nucleic acid amplification reaction. Since mC is not affected by BS treatment, the nucleotide corresponding to mC in this nucleotide sequence is cytosine.

[0055] An "MLH1-n-mC compatible probe" hybridizes to the base sequence corresponding to n-mC in the detection region of the MLH1 gene promoter. In other words, it is a probe for detecting the MLH1 gene promoter where a specific cytosine at the methylation target site is not methylated. The "base sequence corresponding to n-mC" refers to the base sequence corresponding to that n-mC in the amplification product after BS treatment of the MLH1 gene promoter and nucleic acid amplification reaction. On genomic DNA, n-mC undergoes a base conversion of cytosine to uracil upon BS treatment. Therefore, in the nucleic acid amplification product using this as a template, the base corresponding to n-mC in this base sequence becomes thymine.

[0056] When the methylation detection kit of the present invention includes one pair of MLH1-mC-compatible probes and MLH1-n-mC-compatible probes, the MLH1-mC-compatible probes and MLH1-n-mC-compatible probes are configured to hybridize to the nucleotide sequence corresponding to mC or the nucleotide sequence corresponding to n-mC at the same methylation target site. In both probes, the nucleotide sequences and base lengths other than the specific cytosine used to detect the presence or absence of methylation may be the same or different. Furthermore, when the kit includes two or more pairs of MLH1-mC-compatible probes and MLH1-n-mC-compatible probes, in principle, each pair is configured to hybridize to the nucleotide sequence corresponding to mC or the nucleotide sequence corresponding to n-mC at the same methylation target site.

[0057] As described above, the MLH1 probes consist of nucleotide sequences that hybridize to the nucleic acid amplification product in the detection region of the MLH1 gene promoter after BS treatment. The MLH1-mC compatible probe hybridizes to the nucleotide sequence corresponding to mC, and the MLH1-n-mC compatible probe hybridizes to the nucleotide sequence corresponding to n-mC. In this case, the nucleic acid amplification product into which both probes hybridize may be either the sense strand side or the antisense strand side, or both strands. When both probes hybridize to the sense strand side of the nucleic acid amplification product, both probes consist of nucleotide sequences complementary to that sense strand. When both probes hybridize to the antisense strand side of the nucleic acid amplification product, both probes consist of nucleotide sequences complementary to that antisense strand. Furthermore, when both probes hybridize to both the sense strand side and the antisense strand side of the nucleic acid amplification product, the two sets of probes consist of nucleotide sequences complementary to the antisense strand and the sense strand of the nucleic acid amplification product, respectively.

[0058] The methylation target sites that the MLH1 probe detects for the presence or absence of methylation are, as mentioned above, at least one cytosine of a CpG sequence located in the C region and / or D region of the MLH1 gene promoter sequence. In the MLH1 gene promoter sequence shown in Sequence ID No. 1, these cytosines are located at positions 550, 557, 567, 569, 575, 594, 605, and 620 in the C region, and at positions 689, 712, 736, 744, 777, 792, and 812 in the D region. Preferred methylation target sites are positions 557, 567, 569, and 575 in the C region, and positions 736 and 744 in the D region. Particularly preferred methylation target sites are positions 567 and 569 in the C region, and position 744 in the D region. Therefore, the MLH1 probe only needs to be configured to hybridize one of these methylation target sites to a base sequence corresponding to a specific cytosine for which the presence or absence of methylation should be detected.

[0059] As a specific example of an MLH1 probe, if the detection region is the C region and the methylation target sites for detecting the presence or absence of methylation are the cytosines at positions 557, 567, and 569 in the MLH1 gene promoter sequence shown in Sequence ID No. 1, then an MLH1-mC corresponding probe could be, for example, the 27-base polynucleotide shown in Sequence ID No. 4. Furthermore, an MLH1-n-mC corresponding probe that pairs with the MLH1-mC corresponding probe could be, for example, the 20-base polynucleotide shown in Sequence ID No. 5.

[0060] Furthermore, if the detection region is the D region and the methylation target sites for detecting the presence or absence of methylation are the cytosines at positions 736 and 744 in the MLH1 gene promoter sequence shown in Sequence ID No. 1, then an example of an MLH1-mC corresponding probe is a 19-base polynucleotide shown in Sequence ID No. 6. In addition, an example of a 16-base polynucleotide shown in Sequence ID No. 7 is a MLH1-n-mC corresponding probe that pairs with the MLH1-mC corresponding probe.

[0061] The MLH1 probe may contain other nucleotide sequences in addition to the nucleotide sequence complementary to the nucleotide sequence in the nucleic acid amplification product after BS treatment of the detection region of the MLH1 gene promoter. In this case, it is preferable that the other nucleotide sequences are located at the 5' end and / or 3' end of the nucleotide sequence complementary to the nucleic acid amplification product.

[0062] C. Labeling of MLH1 Probes MLH1 probes may have some or all of the phosphate groups, sugars, and / or bases of the nucleotides that constitute them labeled with a labeling substance. The labeling position of the labeling substance on the probe may be determined appropriately according to the characteristics of the labeling substance and the intended use, and is not limited, but the 5' end, 3' end, and / or the SNP site to be detected are preferred. Any substance known in the art can be used as the labeling substance. Examples include DIG, fluorescent substances, quenchers, chemiluminescent substances, radioisotopes, biotin, or magnetic beads. Labeling of nucleotides with each labeling substance can be carried out by known methods.

[0063] The term "fluorescent substance" refers to a substance that becomes excited by absorbing excitation light of a specific wavelength and emits fluorescence when returning to its original ground state. In this specification, fluorescent substances are included. Examples include FITC, Texas, Cy3, IC3, Cy5, IC5, Cy7, IC7, FAM, HEX, VIC, JOE, ROX, TET, Bodipy493, NBD, TAMRA, Quasar® 670, Quasar® 705, CAL Fluor® Red 610, fluorescein or its derivatives, or rhodamine or its derivatives.

[0064] The term "quencher" refers to a substance that absorbs the excitation energy of the fluorescent substance and suppresses its fluorescence. Examples include AMRA, DABCYL, BHQ-1, BHQ-2, or BHQ-3. Generally, quenchers are used in combination with fluorescent substances. Since different types of quenchers have different fluorescence suppression wavelength ranges, when combining a fluorescent substance with a quencher, it is important to use a quencher that can suppress the fluorescence of the fluorescent substance used for labeling. For example, if the fluorescent substance is FAM, HEX, TET, etc., it should be combined with BHQ1, which has a suppression wavelength range of 480 nm to 580 nm. If the fluorescent substance is Cy3, IC3, Cy5, IC5, ROX, TAMRA, Texas, etc., it should be combined with BHQ2, which has a suppression wavelength range of 550 nm to 650 nm. The arrangement of the fluorescent substance and quencher in the probe is not particularly limited, as long as both substances are arranged so that the quencher can suppress the fluorescence produced by the fluorescent substance. For example, one end of the probe (either the 5' or 3' end) can be labeled with a fluorescent substance, and the other end (either the 3' or 5' end) can be labeled with a quencher.

[0065] The aforementioned "chemiluminescent material" refers to a substance that, after being excited by a chemical reaction, releases the difference in energy as light when returning to its ground state. Examples include luminol, rofin, and lucigenin.

[0066] The aforementioned "radioactive isotopes" refer to isotopes with different mass numbers that emit radiation. For example, 32 P, 33 P, or35 S is one example.

[0067] The methylation detection kit of the present invention may contain two or more MLH1 probes.

[0068] (3) Control primer set The "control primer set" consists of one or more primers configured to hybridize to the base sequence of a control nucleic acid and amplify the control region containing n-mC.

[0069] The control region is included in the control nucleic acid and is the target for detection to confirm the success or failure of the reaction after BS treatment. It includes base sequences containing n-mC, particularly base sequence regions containing one or more cytosines other than CpG that are unlikely to be methylated.

[0070] As mentioned above, the control nucleic acid is a nucleic acid containing a control region for confirming the effectiveness of the BS treatment. The type of DNA is not limited as long as it coexists with the MLH1 gene promoter in the sample. For example, within the same genomic DNA in the sample, another gene different from the MLH1 gene or its regulatory region, or a nucleic acid region other than a gene or regulatory region (e.g., a spacer region), or even the MLH1 gene or its promoter can serve as the control nucleic acid. However, if the MLH1 gene promoter is the control nucleic acid, the control region and the detection region must consist of different base sequences. Alternatively, it may be an exogenous polynucleotide added to the sample containing genomic DNA. While not limited, the control nucleic acid is preferably a base sequence region containing another gene different from the MLH1 gene or its regulatory region on the same genome. Housekeeping genes such as the β-actin (ACTB) gene are particularly suitable as the other different gene. One control region is sufficient, but multiple control regions are also acceptable.

[0071] A control primer set can be appropriately designed according to the common methods of the field, such that n-mC is located within the base sequence region (excluding the primer sequence) that is amplified by the primer set. While not limited to this, it is preferable that the region into which the primers hybridize does not contain unmethylated cytosine when designing a control primer set. If the region into which the primers hybridize contains unmethylated cytosine, it is converted to uracil by BS treatment and then to thymine by subsequent quantitative nucleic acid amplification. Therefore, if the control primer set is to hybridize to both strands, two sets are required: one for BS treatment and one for n-BS treatment. However, if the region into which the primers hybridize does not contain unmethylated cytosine, one set of primers is sufficient, regardless of whether BS treatment or n-BS treatment is performed.

[0072] The basic design and configuration (including labeling with a labeling substance) of the Fw primer and Rv primer of the control primer set are the same as those of the Fw primer and Rv primer of the MLH1 primer set, so a detailed explanation is omitted here. Note that in the control primer set used in the examples described later, the R primer (ACT-R) is IC5 labeled.

[0073] Specific examples of control primer sets include, for instance, the Fw primer (ACT-F) shown in SEQ ID NO: 8 and the Rv primer (ACT-R) shown in SEQ ID NO: 9, when the control nucleic acid is the β-actin gene.

[0074] (4) Bisulfite-treated detection probe (BS-treated detection probe) The "bisulfite (BS)-treated detection probe" is one of the control probes and hybridizes to the base sequence corresponding to one or more n-mCs in the BS-treated control region and the amplified product after the nucleic acid amplification reaction. Since all n-mCs in the control region are converted to uracil by BS treatment, in the nucleic acid amplification product using the BS-treated control region as a template, the base corresponding to the n-mC becomes thymine. The BS-treated detection probe hybridizes to the base sequence in the amplified product after BS treatment and the nucleic acid amplification reaction that includes the converted site, for example, the site converted from cytosine to thymine.

[0075] The basic design and configuration (including labeling with labeling material) of the primer set for BS-treated detection probes are the same as those of the MLH1-mC compatible probe described above, so a detailed explanation is omitted here.

[0076] As a specific example of the BS-treated detection probe, if the control nucleic acid is the β-actin gene, a polynucleotide consisting of the base sequence shown in Sequence ID No. 10 can be used, which hybridizes to the amplification product obtained after nucleic acid amplification using the aforementioned control primer set (n-mC-Fw / n-mC-Rv) with the β-actin gene after BS treatment as a template.

[0077] (5) Non-bisulfite-treated detection probe (n-BS-treated detection probe) The "non-bisulfite (n-BS)-treated detection probe" is one of the control probes, and is a probe that is paired with the BS-treated detection probe, and hybridizes to the base sequence corresponding to n-mC in the amplification product after a nucleic acid amplification reaction in which the control region has not been treated with bisulfite.

[0078] If BS treatment is not performed, the n-mC molecules in the control region will not be converted to uracil. Therefore, in nucleic acid amplification products using the control region as a template, all bases corresponding to these n-mC molecules remain cytosine.

[0079] The n-BS-treated detection probe is used in conjunction with the BS-treated detection probe and is configured to hybridize to the base sequence corresponding to the same n-mC contained in the same control region of the same control nucleic acid. However, the base sequences and base lengths other than the specific cytosine to be detected may be the same or different for both probes. Furthermore, when two or more sets of BS-treated detection probes and n-BS-treated detection probes are included, in principle, each set is configured to hybridize to the base sequence corresponding to the same n-mC, or to the base sequence corresponding to the same n-mC.

[0080] A pair of BS-treated detection probes and n-BS-treated detection probes hybridize to the same n-mC-corresponding base sequence in the control region. However, because the base sequences of the nucleic acid amplification product in the BS-treated control region and the nucleic acid amplification product in the untreated control region are different, the BS-treated detection probe can only hybridize to the nucleic acid amplification product in the BS-treated control region, and the n-BS-treated detection probe can only hybridize to the nucleic acid amplification product in the untreated control region. By utilizing this difference, when the nucleic acid amplification product of the BS-treated control nucleic acid is mixed with each probe, it is possible to determine whether the BS treatment was successful or unsuccessful based on which probe was able to hybridize.

[0081] The basic design and configuration (including labeling with a labeling substance) of the primer set for the n-BS-treated detection probe control are the same as those of the MLH1-n-mC-compatible probe described above, so a detailed explanation is omitted here.

[0082] As a specific example of the n-BS treatment detection probe, if the control nucleic acid is a β-actin gene, a polynucleotide consisting of the base sequence shown in Sequence ID No. 11 can be used to hybridize the amplification product obtained after nucleic acid amplification using the aforementioned control primer set (n-mC-Fw / n-mC-Rv) with an untreated β-actin gene as a template. By using the n-BS treatment detection probe shown in Sequence ID No. 11 in pair with the BS treatment detection probe shown in Sequence ID No. 10, it is possible to determine the quality of the BS treatment in the control nucleic acid after BS treatment.

[0083] (6) Detection reagents, etc. In addition to the above components, the methylation detection kit of the present invention may also include, for example, reagents necessary for reverse transcription and nucleic acid amplification reactions (RTase, dNTPs, Taq polymerase, etc.), reagents for detecting amplified nucleic acid fragments (fluorescent / luminescent reagents, etc.), and protocols describing methods for detecting appropriate reaction conditions, etc.

[0084] (7) Microarrays The methylation kit of the present invention may be made into a device having the configuration of a microarray (nucleic acid array, DNA chip).

[0085] For example, one configuration involves arranging and fixing the 5' ends of MLH1 probes (MLH1-mC compatible probes and / or MLH1-n-mC compatible probes), BS processing detection probes, and n-BS processing detection probes in a high-density, small-spot-like manner on a substrate carrier (chip).

[0086] The material of the carrier can be any material known in the art and is not particularly limited. For example, precious metals (including platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, etc.), conductive materials such as carbon (including graphite and carbon fiber), silicon materials (including single-crystal silicon, amorphous silicon, silicon carbide, silicon oxide, silicon nitride, and SOI), inorganic materials (including glass, quartz glass, alumina, sapphire, ceramics, forsterite, photosensitive glass, etc.), and organic materials (polyethylene, ethylene, polypropylene, cyclic polyolefin, polyiso Examples include butylene, polyethylene terephthalate, unsaturated polyester, fluororesin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenolic resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene styrene copolymer, polyphenylene oxide, and polysulfone.

[0087] Although not limited, a carrier having a carbon layer and a chemically modified group on its surface is preferred. Such carriers include those having a carbon layer and a chemically modified group on the surface of the carrier, and those having a chemically modified group on the surface of a carrier composed of a carbon layer.

[0088] The form of the carrier is not particularly limited, but in the case of a microarray, a flat plate structure is preferred. The shape of the carrier is not limited, such as rectangular, square, and round, etc., but usually 1 mm 2 ~75 mm 2 、2 mm 2 ~10 mm 2 、or 3 mm 2 ~5 mm 2 is preferred. From the viewpoint of being easy to manufacture into a flat plate structure, a carrier made of a silicon material or a resin material is preferred, and particularly a carrier having a carbon layer and a chemically modified group on the surface of single crystal silicon is more preferred. [[ID=十六]] [[ID=十七]]

[0089] The carbon layer formed on the carrier is not particularly limited, but it is preferable to use synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (e.g., diamond-like carbon), amorphous carbon, a carbon-based substance (e.g., graphite, fullerene, carbon nanotube), any of their mixtures, or a laminate thereof. Also, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide, etc. may be used. The carbon layer is advantageous in terms of excellent chemical stability and being able to withstand reactions in subsequent introduction of chemically modified groups and binding to the analyte, having flexibility in binding to the analyte by electrostatic binding, being transparent to the detection system UV due to no UV absorption, and being able to conduct electricity during electroblotting.

[0090] Carbon layers can be formed by known methods. Examples include microwave plasma CVD (Chemical vapor deposit), ECRCVD (Electric cyclotron resonance chemical vapor deposit), ICP (Inductive coupled plasma), DC sputtering, ECR (Electric cyclotron resonance) sputtering, ionization evaporation, arc evaporation, laser evaporation, EB (Electron beam) evaporation, and resistance heating evaporation.

[0091] In high-frequency plasma CVD, a glow discharge generated between electrodes by high frequency decomposes the raw material gas (methane), and a carbon layer is synthesized on the support. In ionization evaporation, thermionic electrons generated by a tungsten filament are used to decompose and ionize the raw material gas (benzene), and a carbon layer is formed on the support by a bias voltage. The carbon layer may also be formed by ionization evaporation in a mixed gas consisting of 1 to 99 volume percent hydrogen gas and 99 to 1 volume percent of the remaining methane gas.

[0092] In arc evaporation, a DC voltage is applied between a graphite material (cathode evaporation source) and a vacuum vessel (anode) to induce an arc discharge in a vacuum, generating a plasma of carbon atoms from the cathode. By applying an even more negative bias voltage to the support than that applied to the evaporation source, carbon ions in the plasma are accelerated toward the support, thereby forming a carbon layer.

[0093] In laser deposition, for example, a carbon layer can be formed by irradiating a graphite target plate with pulsed Nd:YAG laser light to melt it and depositing carbon atoms onto a glass support.

[0094] When forming a carbon layer on the support surface, the thickness of the carbon layer is usually around a single molecular layer to 100 μm. If it is too thin, the surface of the underlying substrate may be locally exposed, and if it is too thick, productivity will be poor. Therefore, it is preferably 2 nm to 1 μm, and more preferably 5 nm to 500 nm.

[0095] By introducing chemically modifying groups to the surface of the support on which the carbon layer is formed, each probe can be firmly immobilized on the support. The chemically modifying groups to be introduced can be appropriately selected by those skilled in the art and are not particularly limited, but examples include amino groups, carboxyl groups, epoxy groups, formyl groups, hydroxyl groups, and active ester groups.

[0096] The introduction of amino groups can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas, or by plasma treatment. Alternatively, it can be carried out by chlorinating the carbon layer with ultraviolet light in chlorine gas, and then irradiating it with ultraviolet light again in ammonia gas. Alternatively, it can be carried out by reacting the chlorinated carbon layer with polyhydric amine gases such as methylenediamine and ethylenediamine.

[0097] The introduction of carboxyl groups can be carried out, for example, by reacting a suitable compound with the aminated carbon layer as described above. Examples of compounds used to introduce carboxyl groups include those with formula XR 1- COOH (where X is a halogen atom, R is a halogen atom) 1 Halocarboxylic acids represented by (where represents a divalent hydrocarbon group with 10 to 12 carbon atoms), such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R 2 -COOH (in the formula, R 2 Dicarboxylic acids represented by a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms, such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, and phthalic acid; polycarboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid, and butanetetracarboxylic acid; formula: R 3 -CO-R 4 -COOH (in the formula, R 3 R is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms. 4 Keto acids or aldehyde acids represented by (where represents a divalent hydrocarbon group having 1 to 12 carbon atoms); formula: X-OC-R 5 -COOH (where X is a halogen atom, R is a halogen atom) 5represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms. Examples include monohalides of dicarboxylic acids represented by ), such as succinic acid monolide and malonic acid monolide; and acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

[0098] The introduction of epoxy groups can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the aminated carbon layer as described above. Alternatively, it can be obtained by reacting an organic peracid with the carbon-carbon double bonds contained in the carbon layer. Examples of organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

[0099] The introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the aminated carbon layer as described above.

[0100] The introduction of hydroxyl groups can be carried out, for example, by reacting water with the chlorinated carbon layer as described above.

[0101] Active ester groups refer to esters that have a highly acidic electron-withdrawing group on the alcohol side of the ester group, thereby activating nucleophilic reactions; in other words, ester groups with high reactive activity. These are ester groups that have an electron-withdrawing group on the alcohol side of the ester group and are more active than alkyl esters. Active ester groups are reactive with groups such as amino groups, thiol groups, and hydroxyl groups. More specifically, phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, and esters of heterocyclic hydroxy compounds are known to be active ester groups that have much higher activity than alkyl esters, etc. More specifically, examples of active ester groups include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, and 5-norbornene-2,3-dicarboximide group, with N-hydroxysuccinimide group being particularly preferred.

[0102] The introduction of an active ester group can be carried out, for example, by activating the carboxyl group introduced as described above with a dehydration condensation agent such as cyanamide or carbodiimide (e.g., 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and a compound such as N-hydroxysuccinimide. This process makes it possible to form a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the terminal of the hydrocarbon group via an amide bond (Japanese Patent Application Publication No. 2001-139532).

[0103] In a microarray, the subject's genomic DNA is treated with BS, and then the detection and control regions are amplified by a nucleic acid amplification reaction. The resulting amplified products are then reacted with probes on the microarray. Detection and quantification can be achieved by detecting and measuring fluorescence based on hybridization of target nucleic acids, etc., using a microplate reader or scanner. The presence or absence of methylated cytosine in the C and D regions of the MLH1 gene promoter can be determined from the detected fluorescence intensity ratio and a preset threshold, and the quality of the BS treatment can also be determined using BS-treated detection probes and n-BS-treated detection probes. If the treatment is inadequate, the presence or absence of methylation should not be determined, and the system should be programmed to prompt re-measurement.

[0104] 2. Diagnostic support kit for Lynch syndrome-related tumors 2-1. Overview A second aspect of the present invention is a diagnostic support kit for Lynch syndrome-related tumors (often abbreviated as "diagnostic support kit" in this specification). The diagnostic support kit of the present invention can assist in the definitive diagnosis of Lynch syndrome-related tumors in a subject by detecting methylation or demethylation in the detection region of the MLH1 gene promoter in the subject's genomic DNA using the methylation detection kit of the first aspect.

[0105] As mentioned above, Lynch syndrome-related tumors and sporadic colorectal cancer do not show clinically distinct characteristics, making definitive diagnosis impossible using conventional methods such as MSI testing alone. However, it has become clear that methylation of CpG islands in the MLH1 gene promoter is observed in the genomic DNA of patients with sporadic colorectal cancer, while methylation in the same promoter is not observed in the genomic DNA of patients with Lynch syndrome-related tumors (JG Herman, et al., 1998, Sci. USA, 95: 6870-6875).

[0106] Based on the above findings, it became possible to determine which disease a subject has by detecting the presence or absence of methylation in the MLH1 gene promoter using a sample derived from a subject suspected of having Lynch syndrome-related tumors or sporadic colorectal cancer.

[0107] Generally, the BS method, which utilizes base conversion through BS treatment, is used to detect DNA methylation. While this method can determine the presence or absence of methylation of the base to be detected (cytosine), it cannot make a determination if the BS treatment itself is ineffective, thus posing a problem of misdiagnosis between sporadic colorectal cancer and Lynch syndrome-related tumors.

[0108] In the methylation detection kit of the first embodiment, by performing BS treatment on a control region containing unmethylated cytosine together with the detection region, it becomes possible to accurately determine whether or not the target cytosine in the detection region is methylated. Furthermore, by using the diagnostic support kit of the present invention, it is possible to provide highly accurate and low-misdiagnosis diagnostic support in the diagnosis of sporadic colorectal cancer and Lynch syndrome-related tumors.

[0109] 2-2. Structure The diagnostic aid kit of the present invention is an invention that uses the methylation detection kit of the first embodiment for a specific purpose, namely, as a diagnostic aid to determine whether the disease of a subject is Lynch syndrome-related tumor or sporadic colorectal cancer. Accordingly, the basic structure of the diagnostic aid kit of the present invention is the same as the methylation detection kit of the first embodiment. That is, it includes an MLH1 primer set, an MLH1 probe, a control primer set, a BS-treated detection probe, and an n-BS-treated detection probe as essential components. The specific configuration of each component is described in the first embodiment, so a detailed explanation is omitted here.

[0110] 3. Method for Detecting MLH1 Gene Promoter Methylation 3-1. Overview A third aspect of the present invention is a method for detecting methylation of the MLH1 gene promoter (often abbreviated as "methylation detection method" in this specification). The method for detecting methylation of the MLH1 gene promoter of the present invention allows for the determination of whether or not methylation of the MLH1 gene promoter is occurring, and simultaneously determines the quality of the bisulfite treatment used for methylation detection. This provides a highly accurate and low-false determination result regarding the presence or absence of methylation in the MLH1 gene promoter.

[0111] 3-2. Method The flow chart of the methylation detection method of the present invention is shown in Figures 4 to 6. The flow chart shown in Figure 4 is the main flow chart of the methylation detection method of the present invention, and includes the bisulfite treatment step (S0402), quantitative nucleic acid amplification step (S0403), bisulfite treatment determination step (S0404), and methylation determination step (S0405) as essential steps, and the genomic DNA preparation step (S0401) as an optional step.

[0112] Furthermore, within the quantitative nucleic acid amplification step (S0403) in the main flow shown in Figure 4, two subflows shown in Figures 5 and 6 are processed in parallel. The flow shown in Figure 5 is a flow for performing quantitative nucleic acid amplification on a control region (control region flow), and includes the control region amplification step (S0501), the control probe binding step (S0502), and the control probe measurement step (S0503) as essential steps. The flow shown in Figure 6 is a flow for performing quantitative nucleic acid amplification on a detection region (detection region flow), and includes the detection region amplification step (S0601), the MLH1 probe binding step (S0602), and the MLH1 probe measurement step (S0603) as essential steps.

[0113] The control region flow and the detection region flow are independent flows, and their processing order is not restricted. For example, the control region flow may be performed before the detection region flow, or vice versa. Furthermore, they may be performed simultaneously. In particular, steps with similar operational procedures in both flows can be performed simultaneously. That is, for the control region amplification step (S0501) and the detection region amplification step (S0601) which perform nucleic acid amplification, the control probe binding step (S0502) and the MLH1 probe binding step (S0602) which perform probe binding, and the control probe measurement step (S0503) and the MLH1 probe measurement step (S0603) which perform probe measurement, the primer sets and probes used in each step may be combined and performed simultaneously in one reaction system (in one sample). The following describes each step in detail.

[0114] (1) Genomic DNA preparation process The "genomic DNA preparation process" (S0401) is a process for preparing genomic DNA from a sample derived from the subject.

[0115] In this specification, "subject" refers to an individual subjected to the methylation detection method of the present invention. While not limited to this type, it is generally human. Patients suspected of having Lynch syndrome-related tumors or sporadic colorectal cancer are particularly preferred.

[0116] The type of sample is not restricted. Blood, such as whole blood, is preferred due to its easy availability regardless of the time of collection, low invasiveness to the subject, and ease of handling as a liquid. Other samples may include formalin-fixed paraffin-embedded sections and frozen tissue. ctDNA (circulating tumor DNA) and cfDNA (cell-free DNA) can also be used as samples.

[0117] If the sample is blood, it is acceptable as long as the blood has been collected according to a known method, and there are no particular limitations. For example, blood collected directly by injection from a vein, blood that has been treated with anticoagulation by adding heparin or the like to collected whole blood, and blood that has been stored refrigerated or frozen can all be used as samples.

[0118] The sample volume to be used can be 100 μL to 1 mL, 50 μL to 5 mL, 20 μL to 10 mL, 10 μL to 50 mL, 5 μL to 80 mL, or 1 μL to 100 mL if it is blood.

[0119] The method for extracting genomic DNA from a sample should be prepared according to the standard methods in the relevant field, depending on the type of sample, such as blood or tissue. For example, extraction can be performed according to the nucleic acid extraction method (DNA extraction method) described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Furthermore, various life science manufacturers commercially offer kits for extracting nucleic acids from various samples, which may also be used. When using a kit, the specific extraction method should be carried out according to or in accordance with the attached protocol.

[0120] (2) Bisulfite Treatment Process The "Bisulfite (BS) Treatment Process" (S0402) is a process in which genomic DNA derived from the test subject is treated with BS. In this process, a portion of the sample containing genomic DNA is subjected to BS treatment, and the remaining portion is stored as untreated genomic DNA for subsequent processes.

[0121] The genomic DNA used in this process is, in principle, subject-derived genomic DNA containing the MLH1 gene promoter encompassing the detection region and the control nucleic acid encompassing the control region. However, the control nucleic acid may be exogenous DNA other than subject-derived genomic DNA. In that case as well, in this specification, it shall be mixed with subject-derived genomic DNA and the following steps shall be performed as part of the mixture.

[0122] BS treatment can be carried out using conventional methods in the field. For example, the genomic DNA and bisulfite can be mixed and reacted at a predetermined temperature for a predetermined time. The bisulfite is not limited, but for example, sodium bisulfite (NaHSO3), potassium bisulfite (KHSO3), ammonium bisulfite ((NH4)HSO4), etc. can be used. Sodium bisulfite is preferred. The concentration of bisulfite in the reaction solution used for BS treatment is not particularly limited as long as it is sufficient to adequately convert unmethylated cytosine in the genomic DNA. For example, a final concentration of 1M to 15M, 2M to 10M, or 3M to 6M is acceptable. The reaction conditions, such as reaction temperature and reaction time, and the incubation conditions (temperature and time) can be appropriately set according to the amount of bisulfite added. For example, if bisulfite is added at a final concentration of 6M, incubation at 50 to 80°C for 10 minutes to 7 hours is sufficient.

[0123] After BS treatment, the reaction may be washed to remove bisulfites if necessary. In this step, if the BS treatment is successful, all n-mC contained in the detection region and control region will be replaced with uracil.

[0124] (3) Quantitative nucleic acid amplification step The "quantitative nucleic acid amplification step" (S0403) is a step in which a quantitative nucleic acid amplification reaction is performed using the BS-treated genomic DNA after the BS treatment step and the non-BS-treated genomic DNA, i.e., the genomic DNA before the BS treatment step.

[0125] "Quantitative nucleic acid amplification reaction" refers to the quantitative measurement of the amplification product (amplicon) produced by a nucleic acid amplification reaction.

[0126] "Nucleic acid amplification" refers to a method of amplifying a specific target nucleic acid region sandwiched between Fw / Rv primers by repeatedly performing a nucleic acid extension reaction using nucleic acid polymerase. Examples include PCR (polymerase chain reaction), LAMP (Loop-Mediated Isothermal Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids). PCR is preferred because it is the most widely used method and various application technologies have been developed. For example, qPCR (quantitative PCR) is a known quantitative nucleic acid amplification method using PCR, and various dedicated reagents, kits, and reaction equipment for qPCR are commercially available from various manufacturers.

[0127] PCR reaction conditions vary depending on the base length of the control region to be amplified, the amount of template genomic DNA (non-BS treated genomic DNA and BS treated genomic DNA), the base length and Tm value of the control primers used, the optimal reaction temperature of the nucleic acid polymerase used, and the optimal pH. Therefore, these conditions should be considered and determined appropriately. As an example, a denaturation reaction can be performed at 94-95°C for 5 seconds to 5 minutes, an annealing reaction at 50-70°C for 10 seconds to 1 minute, and an extension reaction at 68-72°C for 30 seconds to 3 minutes. This constitutes one cycle, and this can be repeated for 15-40 cycles, followed by a final extension reaction at 68-72°C for 30 seconds to 10 minutes. When using a commercially available PCR kit, in principle, the protocol provided with the kit should be followed.

[0128] In quantitative nucleic acid amplification reactions, it is desirable to label the amplified product so that it can be identified. Methods for labeling the amplified product are not particularly limited, but include, for example, pre-labeling primers, using labeled nucleotides as substrates in the nucleic acid amplification reaction, and pre-labeling probes that specifically bind to the amplified product. The labeling substance is not particularly limited, but radioactive isotopes, fluorescent substances, or organic compounds such as DIG or biotin can be used.

[0129] In this process, as mentioned above, two independent flows, namely the control region flow shown in Figure 5 and the detection region flow shown in Figure 6, are processed in parallel. The following describes each subflow.

[0130] (3-1) Control Region Flow The control region flow is a flow in which a quantitative nucleic acid amplification reaction is performed on the control region using BS-treated genomic DNA and non-BS-treated genomic DNA as templates. This flow includes a control region amplification step (S0501), a control probe binding step (S0502), and a control probe measurement step (S0503). Each step will be explained below.

[0131] (3-1-1) Control Region Amplification Step The "Control Region Amplification Step" (S0501) is a step in which a control region containing n-mC is amplified using a control primer set capable of amplifying the control region, with non-BS treated genomic DNA, i.e., genomic DNA before the BS treatment step and BS treated genomic DNA after the BS treatment step, as templates.

[0132] The control primer set may be a primer set having the configuration described in "(3) Control Primer Set" in the chapter "1-3. Configuration" of the first embodiment. The method for amplifying the control region is achieved by the nucleic acid amplification method described above.

[0133] Furthermore, the basic conditions for the nucleic acid amplification reaction in this step and the detection region amplification step (S0601) in the detection region flow described later may be the same, except for the difference in the primer sets used. Therefore, although both steps constitute independent flows, they can be performed in the same reaction system. In that case, in the test section using the BS-treated genomic DNA of this step as a template, the nucleic acid amplification reaction can be performed simultaneously using the primer set having the configuration described in "(3) Control Primer Set" in the "1-3. Configuration" section and the primer set having the configuration described in "(1) MLH1 Primer Set".

[0134] (3-1-2) Control probe coupling step The "control probe coupling step" (S0502) is a step in which a control probe is hybridized to the BS-treated control region amplified product and the non-BS-treated control region amplified product (n-BS-treated control region amplified product) after the control region amplification step.

[0135] The control probes include BS-treated detection probes and n-BS-treated detection probes. In principle, the BS-treated detection probe is hybridized to the BS-treated control region amplification product, and the n-BS-treated detection probe is hybridized to the n-BS-treated control region amplification product. However, if necessary, the non-BS-treated detection probe may be hybridized to the BS-treated control region amplification product, and the BS-treated detection probe may be hybridized to the n-BS-treated control region amplification product.

[0136] Each control probe may be one having the configuration described in "(4) Bisulfite-treated detection probe (BS-treated detection probe)" and "(5) Non-bisulfite-treated detection probe (n-BS-treated detection probe)" in the chapter "1-3. Configuration" of the first embodiment.

[0137] The coupling reaction between the BS-treated control region amplified product and the BS-treated detection probe, and the coupling reaction between the n-BS-treated control region amplified product and the n-BS-treated detection probe are carried out independently.

[0138] The hybridization reaction, in which a control probe is bound to the amplified product, is not particularly limited as long as the control probe, which has a base sequence complementary to the control region contained in the amplified product, can be bound. However, it is preferable to carry it out under stringent conditions. "Stringent conditions" refer to conditions under which nonspecific hybridization does not occur or is unlikely to occur. Specifically, for example, this refers to conditions in which the hybridization reaction is carried out at 50°C for 16 hours, followed by washing in 2×SSC / 0.2% SDS, 5×Denhardt reagent, and 100 μg / mL denatured fragmented DNA (e.g., salmon sperm DNA: ssDNA) at 25°C for 10 minutes and then with 2×SSC at 25°C for 5 minutes. It may also be carried out under even more stringent conditions. "Highly stringent conditions" refer to more stringent hybridization reaction conditions, which involve performing hybridization and washing under low salt concentration and / or high temperature conditions. Specifically, this refers to conditions such as incubating the probe with 6×SSC / 0.5% SDS, 5×Denhardt reagent, and 100μg / mL ssDNA at 65°C to 68°C, and then washing in a 2×SSC / 0.1% SDS washing solution, starting at room temperature, gradually reducing the salt concentration in the washing solution to 0.1×SSC and raising the temperature to 68°C until no background signal is detected.

[0139] In the BS-treated control region amplification product obtained after the control region amplification step (S0501) of the BS-treated control nucleic acid, if the BS treatment was successful, the base corresponding to n-mC in the control region is substituted with T. In this case, a BS-treated detection probe having a base sequence complementary to the base sequence of the control region after the base substitution can specifically bind. On the other hand, if the BS treatment failed, the base corresponding to n-mC in the control region remains C. Therefore, in this case, the BS-treated detection probe cannot specifically bind, and the amount of probe bound decreases.

[0140] In the n-BS-treated control region amplification product obtained after the control region amplification step (S0501) using the control nucleic acid before BS treatment, no base substitution occurs due to BS treatment, so the base corresponding to n-mC in the control region remains C. Therefore, an n-BS-treated detection probe having a base sequence complementary to the base sequence of the BS-untreated control region can bind specifically.

[0141] As described above, in this process, the quality of the BS treatment in the BS treatment step (S0402) can be expressed as the difference in the coupling amount between each amplified product after the control region amplification step (S0501) and the control probe.

[0142] Although this step and the MLH1 probe coupling step (S0602) in the detection region flow described later constitute independent flows, the probe coupling conditions in both steps may be the same, except for the difference in the probes used. Therefore, both steps can be carried out in the same reaction system. For example, as described in the control region amplification step (S0501), if the control region amplification step (S0501) and the detection region amplification step (S0601) in the detection region flow described later are carried out in the same reaction system, then this step and the MLH1 probe coupling step (S0602) can be carried out in the same reaction system. In that case, when carrying out this step, two types of probes having the configuration described in "(2) MLH1 probe" in the "1-3. Configuration" chapter of the first embodiment, namely the MLH1-mC compatible probe and the MLH1-n-mC compatible probe, should be added simultaneously to the test section to which the BS-treated detection probe is coupled to the BS-treated control region amplification product.

[0143] (3-1-3) Control probe measurement process The "control probe measurement process" (S0503) is a process of detecting and measuring the amount of each control probe bound to the BS-treated control region amplified product and the n-BS-treated control region amplified product after the control probe binding process (S0502).

[0144] In the control probe binding step (S0502), the control probe was bound to each amplification product by a hybridization reaction. Several methods with different detection means are known for measuring the amount of probe bound based on the hybridization reaction. Examples include Southern blot hybridization, microarray method, surface plasmon resonance method, or quartz crystal microbalance method.

[0145] A. Southern blot hybridization method The Southern blot hybridization method is a method in which DNA amplification products (in this specification, BS-treated control region amplification products or n-BS-treated control region amplification products) that have undergone a nucleic acid amplification process are separated based on base length size by electrophoresis using an agarose gel or polyacrylamide gel, etc., and after blotting onto a filter, they are detected using a probe having a base sequence specific to the target nucleic acid (in this specification, control region amplification products containing n-mC).

[0146] The control probe may be pre-labeled with various labeling substances as described in "(2) MLH1 Probe; C. Labeling of MLH1 Probe" in the "1-3. Composition" section of the first embodiment. This allows for the measurement of the amount of target nucleic acid bound to the DNA amplification product. Alternatively, the primer may be pre-labeled, and the amount of binding between the amplification product containing the labeling substance and the control probe may be measured.

[0147] The amount of binding between the probe based on the label and the amplified product can be measured using measuring devices such as a fluorescence scanner, chemiluminescence imaging analyzer, imaging analyzer, or scintillation counter. For example, when a fluorescent substance is used as the label, the amount of binding can be obtained as a measured value by detecting the fluorescence signal using a fluorescence scanner and quantifying the signal intensity using image analysis software.

[0148] In this specification, "measured value" refers to a value indicating the amount of each probe bound to the target nucleic acid amplification product, as measured in this step and in the MLH1 probe measurement step (S0603) described later. The measured value may be an absolute value such as volume, weight, or number of probes, or a relative value such as concentration, ionic strength, absorbance, or fluorescence intensity.

[0149] B. Microarray Method The "microarray method," as explained in "(7) Microarray" in the chapter "1-3. Configuration" of the first aspect, involves densely arranging and immobilizing probes having a base sequence complementary to all or part of the base sequence of the target nucleic acid on a substrate in a spot-like manner, reacting a sample containing the target nucleic acid with these probes, and detecting and measuring the nucleic acid hybridized to the spots using fluorescence or the like.

[0150] The detection and quantification of target nucleic acids can be performed in essentially the same manner as the Southern blot hybridization method described above. For example, by pre-labeling primers, an amplified product containing the labeling substance can be obtained. This amplified product specifically binds to a fixed probe, and the label on the amplified product can be detected and measured using a microplate reader or fluorescence scanner, thereby achieving detection of the target nucleic acid. Furthermore, by quantifying the labeling intensity using image analysis software, the amount of binding between the control probe and the amplified product can be obtained as a measured value.

[0151] C. Surface Plasmon Resonance Method The "Surface Plasmon Resonance (SPR) method" is a method for highly sensitively detecting and quantifying adsorbents on the surface of a metal thin film by utilizing the surface plasmon resonance phenomenon, in which the reflected light intensity is significantly attenuated at a specific incident angle (resonance angle) when the incident angle of laser light irradiated onto a metal thin film is changed. In this invention, for example, a control probe is immobilized on the surface of the metal thin film, and other parts of the metal thin film surface are blocked. Then, a DNA amplification product (in this specification, a BS-treated control region amplification product or an n-BS-treated control region amplification product) is passed over the metal thin film surface to capture the amplification product that has specifically bound to the control probe via base pairing. A hybrid of the control probe and the DNA amplification product can be detected and measured from the difference in measurement values ​​before and after sample flow. Measurement by surface plasmon resonance can be performed, for example, using an SPR sensor commercially available from Biacore.

[0152] D. Quartz Crystal Microbalance Method The "Quartz Crystal Microbalance (QCM) method" is a mass measurement method that quantitatively detects minute amounts of adsorbed material by measuring the change in resonance frequency, utilizing the phenomenon that when a substance is adsorbed onto the electrode surface attached to a quartz crystal, the resonance frequency of the quartz crystal decreases in proportion to the mass of the adsorbed substance. Similar to the SPR method, detection and quantification using this method can be performed using a commercially available QCM sensor, for example, by base pairing with a control probe fixed to the electrode surface to detect and measure the target nucleic acid in the amplified product after the control region amplification step (S0501).

[0153] Various nucleic acid quantification kits based on the above methods are commercially available from various life science manufacturers and can also be used.

[0154] Although this process and the MLH1 probe measurement process (S0603) in the detection region flow described later constitute independent flows, the measurement conditions in both processes may be the same as long as the binding of the amplified product to the probe in each process can be identified. Therefore, both processes can be carried out in the same reaction system. For example, by using different labeling substances in each flow, the amount of binding between the amplified product and the probe in each flow can be measured based on the difference in labeling substances, even when carried out in the same reaction system.

[0155] (3-2) Detection Region Flow The detection region flow is a flow in which a quantitative nucleic acid amplification reaction is performed on the detection region using BS-treated genomic DNA as a template. This flow includes a detection region amplification step (S0601), an MLH1 probe binding step (S0602), and an MLH1 probe measurement step (S0603). Each step will be explained below.

[0156] (3-2-1) Detection Region Amplification Step The "Detection Region Amplification Step" (S0601) is a step in which the detection region, which includes predetermined methylation target sites in the C region and / or D region, is amplified using the BS-treated genomic DNA as a template, with an MLH1 primer set capable of amplifying the detection region.

[0157] The MLH1 primer set can be any primer set having the configuration described in "(1) MLH1 Primer Set" in the chapter "1-3. Configuration" of the first embodiment.

[0158] The detection region is amplified by nucleic acid amplification. This nucleic acid amplification method and its reaction conditions should be carried out in accordance with the control region amplification step (S0501) described above. Furthermore, as mentioned above, this step and the control region amplification step (S0501) in the control region flow described above can be carried out in the same reaction system.

[0159] (3-2-2) MLH1 probe coupling step The "MLH1 probe coupling step" (S0602) is a step in which the MLH1 probe is hybridized to the detection region amplification product after the detection region amplification step (S0601).

[0160] The MLH1 probe consists of an MLH1-mC-compatible probe and an MLH1-n-mC-compatible probe, as described in "(2) MLH1 Probe" in the chapter "1-3. Composition". The MLH1-mC-compatible probe specifically hybridizes to the nucleotide sequence corresponding to mC in the detection region of the MLH1 gene promoter, and the MLH1-n-mC-compatible probe specifically hybridizes to the nucleotide sequence corresponding to n-mC in the detection region of the MLH1 gene promoter.

[0161] The MLH1 probe used in this process may be one of two types of probes (MLH1-mC compatible probe and MLH1-n-mC compatible probe) having the configuration described in "(2) MLH1 Probe" in the "1-3. Configuration" section of the first embodiment. Either probe can be used in this process.

[0162] The hybridization reaction to bind the MLH1 probe to the detection region amplification product may be carried out in accordance with the method described in the control probe binding step (S0502).

[0163] In this process, the binding reaction between the detection region amplification product and the MLH1-mC corresponding probe, and the binding reaction between the detection region amplification product and the MLH1-n-mC corresponding probe, may be carried out independently or simultaneously in the same reaction system. Furthermore, as mentioned above, this process and the control probe binding process (S0502) in the control region flow described above can be carried out in the same reaction system.

[0164] (3-2-3) MLH1 probe measurement process The "MLH1 probe measurement process" (S0603) is a process for measuring the amount of each probe bound after the MLH1 probe binding process.

[0165] In the MLH1 probe binding step (S0602), similar to the control probe binding step (S0502), each MLH1 probe was bound to the detection region amplification product by a hybridization reaction. In this step as well, the amount of probe bound based on the hybridization reaction can be measured using methods such as Southern blot hybridization, microarray, surface plasmon resonance, or quartz crystal microbalance, based on the hybridization reaction described in the control probe binding step (S0502). Therefore, this step can also be measured using a method similar to that of the control probe binding step (S0502).

[0166] (4) Bisulfite Treatment Determination Step The "bisulfite (BS) treatment determination step" (S0404) is a step in which the quality of the BS treatment in the BS treatment step (S0402) (whether or not the BS reaction was normal) is determined based on the binding ratio of the BS treatment detection probe and the n-BS treatment detection probe obtained after the control probe measurement step (S0503) in the control region flow of the quantitative nucleic acid amplification step (S0403).

[0167] While the determination method based on the binding ratio is not limited, for example, a determination value can be calculated using a BS processing determination formula, and the determination can be made based on that value.

[0168] The BS processing determination formula can be expressed, for example, as follows: (BS processing determination formula) BS processing determination value = n-BS processing detection probe coupling amount / (n-BS processing detection probe coupling amount + BS processing detection probe coupling amount)

[0169] In the above formula, "BS-treated detection probe binding amount" is a measured value indicating the amount of BS-treated detection probe bound to the BS-treated control region amplified product obtained in the control probe measurement step (S0503). In the above formula, "n-BS-treated detection probe binding amount" is a measured value indicating the amount of n-BS-treated detection probe bound to the n-BS-treated control region amplified product obtained in the control probe measurement step (S0503).

[0170] According to this BS processing judgment formula, a large judgment value (close to 1) is obtained if the BS processing is poor, and a small judgment value (close to 0) is obtained if the BS processing is good.

[0171] By substituting the respective measured values ​​into the BS processing judgment formula, a judgment value can be calculated. Based on the obtained judgment value, the quality of the BS processing in the BS processing step (S0402) can be determined.

[0172] The determination method based on the judgment value is not limited to this method, but examples include a method in which a cutoff value is set in advance and the quality of the BS treatment is determined based on that cutoff value, and a method in which the quality of the BS treatment is determined based on the statistically significant difference when comparing the judgment value obtained in this process with the judgment value calculated based on a standard sample in which the BS treatment was measured in advance and was good.

[0173] A "cutoff value" is a boundary value used to classify quantitative results into two groups. For example, a predetermined value can be set as the cutoff value. If the judgment value is greater than or equal to that value, it can be determined that there is a high probability that the BS processing in the BS processing step (S0402) is poor. Conversely, if the judgment value is lower than that value, it can be determined that there is a high probability that the BS processing in the BS processing step (S0402) is good.

[0174] There are no particular limitations on the method for setting the cutoff value. The cutoff value may be determined based on values ​​measured in the same control region, or it may be determined based on a value expected from the number of methylated and unmethylated cytosines in the control region. For example, based on the relationship between the proportion of methylated nucleic acids and the judgment value derived by a similar method, the group of judgment values ​​can be classified by percentile, and the percentile value used for that classification can be set as the cutoff value. Specifically, for example, if the 5th percentile of the judgment value is set as the cutoff value in advance, and values ​​above that value are defined as poor and values ​​below that value are defined as good, then if the judgment value calculated by the newly implemented methylation detection method is less than the 5th percentile, it can be determined that the BS treatment in the BS treatment step (S0402) of that method is likely to be good.

[0175] The method of determination based on "statistical significance" is not limited to any known test method capable of determining the presence or absence of significance. Examples include the t-test and multiple comparison tests. "Statistically significant" means that, under the null hypothesis, the randomness of the obtained value is statistically low and it is meaningful, and "significant difference" means that there is a significant difference between the two values. Specifically, this includes cases where the significance level is less than 5%, 1%, 0.3%, 0.2%, or 0.1%.

[0176] In this process, if the average judgment value calculated based on standard samples in which the BS treatment was successful and the judgment value of the subject are statistically processed using the t-test method or the like, and there is no statistically significant difference between the two, then it can be determined that the BS treatment in the BS treatment process (S0402) was successful. On the other hand, if a significant difference is found, it can be determined that there is a high possibility that the BS treatment was unsuccessful. "Average judgment value calculated based on standard samples in which the BS treatment was successful" refers to the average value of judgment values ​​obtained from multiple standard samples in which the BS treatment was confirmed to be successful using the same method as the methylation detection method performed on the subject.

[0177] (5) Methylation determination step The "methylation determination step" (S0405) is a step performed when the result of the BS treatment determination step (S0404) is judged as good (the BS reaction was normal), and is a step in which the presence or absence of methylation at a predetermined methylation target site is determined based on the binding amount ratio of an MLH1-mC corresponding probe hybridized to a predetermined methylation target site obtained after the MLH1 probe measurement step (S0603) and an MLH1-n-mC corresponding probe hybridized to the same site in the detection region flow of the quantitative nucleic acid amplification step (S0403).

[0178] While the determination method based on the bond amount ratio is not limited, for example, a determination value can be calculated using a methylation determination formula, and the determination can be made based on that value.

[0179] The methylation determination formula can be expressed, for example, as follows: (Methylation determination formula) Methylation determination value = Amount of probe bound to MLH1-mC / (Amount of probe bound to MLH1-mC + Amount of probe bound to MLH1-n-mC)

[0180] In the above formula, "MLH1-mC compatible probe binding amount" is a measured value indicating the amount of MLH1-mC compatible probe bound to the detection region amplification product obtained in the MLH1 probe binding step (S0602). In the above formula, "MLH1-n-mC compatible probe binding amount" is a measured value indicating the amount of MLH1-n-mC compatible probe bound to the detection region amplification product obtained in the MLH1 probe binding step (S0602).

[0181] According to this methylation determination formula, a large (close to 1) determination value is obtained if methylation is present, and a small (close to 0) determination value is obtained if methylation is not present.

[0182] By substituting the measured values ​​into the methylation determination formula, a determination value can be calculated. Based on the obtained determination value, it is possible to determine whether or not methylation has occurred at the methylation target site in the MLH1 gene promoter on the genomic DNA.

[0183] The determination method based on the aforementioned determination value is not limited, but basically it should be carried out in accordance with the method described in the BS treatment determination step (S0404) above. For example, this could involve setting a cutoff value in advance and determining the presence or absence of methylation of the methylation target site based on that cutoff value, or determining the presence or absence of methylation of the methylation target site based on the statistically significant difference when comparing the determination value obtained in this step with the determination value calculated based on a previously measured methylation standard sample.

[0184] When making a determination based on a cutoff value, a predetermined value is set as the cutoff value. If the determination value is greater than or equal to that value, it can be determined that the methylation target site in the C region and / or D region of the MLH1 gene promoter is methylated. Conversely, if the determination value is lower than that value, it can be determined that there is a high probability that the methylation target site is not methylated.

[0185] The method for setting the cutoff value is not particularly limited, but similar to the BS processing determination step (S0404) described above, the determination value group can be classified by percentile based on the relationship between the methylation rate obtained in this step and the determination value, and the percentile value used for that classification can be set as the cutoff value. Specifically, for example, if the 5th percentile of the determination value is set in advance as the cutoff value, and values ​​above that value are defined as methylated and values ​​below that value are defined as unmethylated, then if the determination value calculated by the newly implemented methylation detection method is above the 5th percentile, it can be determined that there is a high probability that the methylation target sites in the C region and / or D region of the MLH1 gene promoter are methylated on the genomic DNA of that subject.

[0186] In the case of this process, the method of determination based on "statistical significance" involves statistically processing the average judgment value calculated based on a standard sample in which the methylation target site on the genomic DNA is methylated, and the subject's judgment value, using the t-test method or the like. If there is no statistically significant difference between the two, it can be determined that the methylation target site is highly likely to be methylated. On the other hand, if a significant difference is observed, it can be determined that the methylation target site is highly likely to be unmethylated.

[0187] <Example 1: Verification of the quality of the bisulfite (BS) reaction in the control region of template DNA> (Objective) To verify that in the MLH1 gene promoter methylation detection method of the present invention, the quality of the BS treatment can be determined without being affected by the presence or absence of cytosine methylation in the control region of template DNA.

[0188] (Methods) The control region was defined as the region containing cytosine (nucleotide sequence: CA) that is unaffected by methylation in the β-actin (ACTB) gene. PCR was performed using pre-BS treatment (n-BS treatment) DNA and bisulfite (BS) treated DNA as templates. The amplified products were hybridized onto chips immobilized with various detection probes, and the fluorescence intensity was detected before calculating the result.

[0189] BS treatment was performed using the EZ DNA Methylation-Lightning Kit (ZYMO RESEARCH) according to the attached protocol.

[0190] For the control region amplification primer set, we used a control region amplification Fw (ACT-F) primer consisting of the nucleotide sequence shown in SEQ ID NO: 8 and a control region amplification Rv (ACT-R) primer consisting of the nucleotide sequence shown in SEQ ID NO: 9. The ACT-R primer was labeled with IC5. PCR amplification was performed according to standard procedures. PCR cycles consisted of 40 cycles, each consisting of 2 minutes at 94°C, followed by 10 seconds at 98°C, 30 seconds at 57°C, and 30 seconds at 68°C, with the temperature maintained at 4°C until the end.

[0191] DNA chips, each immobilized with an n-BS detection probe (SEQ ID NO: 11) corresponding to the control region amplification product before BS treatment (n-BS treatment) and a BS detection probe (SEQ ID NO: 10) corresponding to the control region amplification product after BS treatment, were set in a gene analysis instrument (BIOSHOT HT-32; Toyo Kohan Co., Ltd.) and hybridization reactions were performed with the PCR amplification products.

[0192] After the hybridization reaction, the DNA chip needle, to which the DNA chip was immobilized, was immersed in a washing solution tank to wash the DNA chip. After washing, the DNA chip needle was immersed in a detection solution tank, and a 640 nm single-wavelength laser was irradiated, and the excitation light was captured for 2 seconds with a CCD camera.

[0193] The fluorescence intensity values ​​of each measured sample were used to calculate the judgment value by substituting them into the following formula: [Judgment Value] = [Fluorescence Intensity of n-BS Probe] / ([Fluorescence Intensity of BS Probe] + [Fluorescence Intensity of n-BS Probe])

[0194] (Results) The results are shown in Table 1.

[0195]

[0196] In the table, No. 1 shows the judgment value calculated from the fluorescence intensity when the n-mC region before BS treatment (n-BS treatment) is detected using either the n-BS treatment detection probe (n-mC / n-BS detection probe) or the BS treatment detection probe (n-mC / BS detection probe).

[0197] Furthermore, No. 2 shows the judgment value calculated from the fluorescence intensity when the n-mC region after BS treatment is detected using either an n-BS treatment detection probe (n-mC / n-BS detection probe) or a BS treatment detection probe (n-mC / n-BS detection probe).

[0198] From the results above, in sample No. 1 before BS treatment, the judgment value was close to 1, whereas in sample No. 2 after BS treatment, the judgment value was close to 0 regardless of the presence or absence of cytosine methylation. This result suggests that in the MLH1 gene promoter methylation detection method of the present invention, the control region of the template DNA can determine only the quality of the BS treatment without being affected by the presence or absence of cytosine methylation.

[0199] <Example 2: Determination of MLH1 gene promoter methylation using judgment values> (Objective) To confirm that the MLH1 gene promoter methylation detection method of the present invention can determine whether or not the MLH1 gene promoter is methylated in genomic DNA, and to further verify whether it is possible to make a determination according to the percentage of methylation of the promoter.

[0200] (Methods) Using human genomic DNA derived from peripheral blood, and plasmid DNA as a control nucleic acid containing the C and D regions of the human MLH1 gene promoter and the ACTB gene as templates, the mC and n-mC regions contained in each template were adjusted so that the proportion of mC was between 0% and 100%, and then mixed.

[0201] PCR was performed on the detection region and the control region of the above template using primer sets capable of amplifying each region, and amplified products were obtained. For amplification of the detection region C, the primer set consisted of an Fw primer with the nucleotide sequence shown in SEQ ID NO: 12 and an Rv primer with the nucleotide sequence shown in SEQ ID NO: 13, which was labeled with IC5. For amplification of the detection region D, the primer set consisted of an Fw primer with the nucleotide sequence shown in SEQ ID NO: 14, which was labeled with IC5, and an Rv primer with the nucleotide sequence shown in SEQ ID NO: 15. Furthermore, for amplification of the control region containing the n-mC of the ACTB gene, the primer set consisted of an Fw primer with the nucleotide sequence shown in SEQ ID NO: 8 and an Rv primer with the nucleotide sequence shown in SEQ ID NO: 9, which was labeled with IC5. The PCR conditions were the same as in Example 1.

[0202] Next, DNA chips immobilized with the following probes were placed in a gene analysis device (BIOSHOT HT-32; Toyo Kohan Co., Ltd.): the MLH1-mC probe shown in SEQ ID NO: 4, which corresponds to the mC region amplification product of the C region; the MLH1-n-mC probe shown in SEQ ID NO: 5, which corresponds to the n-mC region amplification product of the C region; the MLH1-mC probe shown in SEQ ID NO: 6, which corresponds to the mC region amplification product of the D region; the MLH1-n-mC probe shown in SEQ ID NO: 7, which corresponds to the n-mC region amplification product of the D region; the BS treatment detection probe shown in SEQ ID NO: 10, which corresponds to the BS treatment control region amplification product; and the n-BS treatment detection probe shown in SEQ ID NO: 11, which corresponds to the non-BS treatment control region amplification product. Hybridization reactions were then performed with the amplification products obtained by PCR.

[0203] After the hybridization reaction, the DNA chip needle, to which the DNA chip was immobilized, was immersed in a washing solution tank to wash the DNA chip. After washing, the DNA chip needle was immersed in a detection solution tank, and a 640 nm single-wavelength laser was irradiated, and the excitation light was captured for 2 seconds with a CCD camera.

[0204] The fluorescence intensity values ​​of each measured sample were introduced into the formula described in Example 1 to calculate the judgment value.

[0205] (Results) Figure 7 shows the results. This figure plots the relationship between the methylation rate in the MLH1 gene promoter and the judgment value. A represents the results for the promoter region C, and B represents the results for the promoter region D.

[0206] In regions C and D, fluorescence intensity and threshold values ​​corresponding to the methylation rate were obtained for both genomic DNA (○) and plasmid DNA (●). The results clearly showed that in both regions, 0% and 5% methylation rates could be clearly separated using a threshold value of 0.1. This suggests that, using the detection method of the present invention, the presence or absence of methylation in a sample can be determined by pre-setting a cutoff value for the threshold value. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A methylation detection kit for the MLH1 gene promoter, comprising: an MLH1 primer set for amplifying a detection region containing a methylation target site of the MLH1 gene promoter; an MLH1 probe that hybridizes to a nucleotide sequence containing the methylation target site in the amplified product of the detection region after bisulfite (BS) treatment and nucleic acid amplification reaction; a control primer set for amplifying a control region containing unmethylated cytosine (n-mC) of a control nucleic acid; a BS-treated detection probe that hybridizes to a nucleotide sequence corresponding to n-mC in the amplified product of the control region after BS treatment and nucleic acid amplification reaction; and a non-bisulfite (n-BS) treated detection probe that hybridizes to a nucleotide sequence corresponding to n-mC in the amplified product of the control region after nucleic acid amplification reaction without bisulfite treatment, wherein the detection region is all or part of the C region and / or D region of the MLH1 gene promoter.

2. The methylation detection kit according to claim 1, wherein the MLH1 gene promoter consists of the nucleotide sequence shown in Sequence ID No.

1.

3. The methylation detection kit according to claim 1, wherein the C region and D region consist of the base sequences shown in Sequence ID No. 2 and 3, respectively.

4. The methylation detection kit according to claim 1, wherein the MLH1 probe comprises an MLH1-mC compatible probe that hybridizes to a base sequence corresponding to methylated cytosine (mC) at a methylation target site, and / or an MLH1-n-mC compatible probe that hybridizes to a base sequence corresponding to unmethylated cytosine (n-mC).

5. The methylation detection kit according to claim 1, comprising a microarray on which the MLH1 probe, the BS treatment detection probe, and the n-BS treatment detection probe are immobilized.

6. The methylation detection kit according to claim 1, wherein the control nucleic acid is the β-actin (ACTB) gene.

7. The methylation detection kit according to claim 6, wherein the BS-treated detection probe includes the nucleotide sequence shown in SEQ ID NO: 10, and the n-BS-treated detection probe includes the nucleotide sequence shown in SEQ ID NO:

11.

8. A diagnostic aid kit for Lynch syndrome-related tumors using the methylation detection kit described in claim 1.

9. A method for detecting methylation of the MLH1 gene promoter, comprising: a BS treatment step of treating genomic DNA derived from a test subject with BS; a control region amplification step of amplifying a control region containing n-mC using a control primer set capable of amplifying the control region, using n-BS treated genomic DNA before the BS treatment step and BS treated genomic DNA after the BS treatment step as templates; a control probe binding step of hybridizing a BS-treated detection probe to the BS-treated control region amplification product and an n-BS-treated detection probe to the non-BS-treated control region amplification product after the control region amplification step; a control probe measurement step of measuring the amount of each probe bound after the control probe binding step; a detection region amplification step of amplifying a detection region containing predetermined methylation target sites in the C region and / or D region using a primer set capable of amplifying the detection region, using the BS-treated genomic DNA as a template; and an MLH1 probe binding step of hybridizing an MLH1-mC-corresponding probe and an MLH1-n-mC-corresponding probe to the detection region amplification product after the detection region amplification step. The method includes: an MLH1 probe measurement step for measuring the amount of each probe bound after the MLH1 probe binding step; a BS processing determination step for determining whether the BS processing is good or bad based on the binding ratio of the BS processing detection probe and the n-BS processing detection probe after the control probe measurement step; and a methylation determination step for determining whether methylation has occurred at a predetermined methylation target site, when the result of the BS processing determination step is deemed good, based on the binding ratio of the MLH1-mC corresponding probe that hybridizes to a predetermined methylation target site after the MLH1 probe measurement step and the MLH1-n-mC corresponding probe that hybridizes to the same site.

10. The method according to claim 9, wherein the determination in the BS processing determination step is made by determining that the BS processing is good if the determination value calculated from the coupling amount of the BS processing detection probe and the coupling amount of the n-BS processing detection probe using a BS processing determination formula is higher than a preset cutoff value, and the BS processing determination formula is expressed as: BS processing determination value = n-BS processing detection probe coupling amount / (n-BS processing detection probe coupling amount + BS processing detection probe coupling amount).

11. The method according to claim 9 or 10, wherein the determination in the methylation determination step is made by determining that a predetermined methylation target site is methylated on genomic DNA if the determination value calculated from the amount of the MLH1-mC corresponding probe bound and the amount of the MLH1-n-mC corresponding probe bound using a methylation determination formula is higher than a preset cutoff value, and the methylation determination formula is expressed as: Methylation determination value = Amount of MLH1-mC corresponding probe bound / (Amount of MLH1-mC corresponding probe bound + Amount of MLH1-n-mC corresponding probe bound).

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