WO2022149575A1 - Kit for detecting mlh1 methylation modification and braf mutation - Google Patents

Abstract

The present invention accurately detects methylation of the MLH1 gene and a spontaneous mutation (V600E) in the BRAF gene. A step for treating sample genome DNA with bisulfite, wherein a region for detecting methylation of the MLH1 gene and a region which includes the V600E spontaneous mutation site in the BRAF gene are each amplified by using a pair of primers, and the region for detecting methylation of the MLH1 gene and the region which includes the V600E spontaneous mutation site in the BRAF gene which are contained in the amplicon are detected by using a probe.

Description

Kit for detecting MLH1 methylation modifications and BRAF mutations

The present invention relates to a kit for detecting methylation modification of the MLH1 gene encoding a DNA mismatch repair protein and a V600E gene mutation in BRAF (v-raf murine sarcoma viral oncogene homolog B1).

Most colorectal cancers are thought to develop due to the accumulation of gene mutations in colorectal mucosal cells, mainly due to exposure to environmental factors (sporadic colorectal cancer). Therefore, the prevalence of sporadic colorectal cancer increases with age. On the other hand, some colorectal cancers are familial and are collectively called hereditary colorectal cancer. In hereditary colorectal cancer, the germline mutation that causes it is found to be a mutation in the tumor suppressor gene or mismatch repair gene, and it is a premature onset, and has the characteristic of developing multiple colorectal cancer and cancer in other organs at a high rate. Among hereditary colorectal cancers, Lynch syndrome (sometimes referred to as hereditary non-polyposis colorectal cancer: HNPCC), which frequently develops colorectal cancer in an autosomal dominant manner, Includes familial adenomatous polyposis (FAP). FAP, which usually develops 100 or more adenomas in the colorectal mucosa, is often easily diagnosed due to its clinical characteristics, while Lynch syndrome, which has no difference in clinical characteristics from sporadic colorectal cancer, is used in daily practice. It is highly likely that many have been overlooked. Reliable identification of hereditary tumors reliably aids in cancer prevention and early detection in the family.

Lynch syndrome is an autosomal dominant disorder mainly caused by germline mutations in mismatch repair genes. Currently, Lynch syndrome is diagnosed according to the following procedure. First, for patients with suspected Lynch syndrome, we will collect clinical information such as family history, age of onset, related cancers, and histopathological features, and consider whether they meet the so-called Amsterdam Standard II or the revised Zebesda Guidelines. If these Amsterdam Standard II or revised Zebesda Guidelines are met, a microsatellite instability (MSI) test of the tumor tissue or an immunohistological test for the causative mismatch repair protein is performed, and the frequency is high. Confirm the disappearance of mismatch repair proteins by MSI (high-frequency MSI: MSI-H) or immunostaining.

Here, microsite instability (MSI) means that microsatellite, which is a repeating sequence of one to several bases existing in the genome due to an abnormality in the mismatch repair mechanism, has a different number of repetitions from normal cells. The phenomenon shown. Frequent microsatellite instability (MSI-H) is often found in tumor tissue in Lynch syndrome. The Amsterdam Standard II may miss Lynch Syndrome due to its strict standards, and the revised Zebesda Guidelines have been devised to make up for its shortcomings. The revised Zebesda Guidelines recommend that MSI tests be performed when 1 to 5 items of clinical information and findings are met. And if the result of the MSI test on the tumor cells is MSI-H, Lynch syndrome is strongly suspected.

A definitive diagnosis of Lynch syndrome is made by identifying pathogenic mutations in the germline of the mismatch repair gene. Specifically, the patient's blood is used to directly test for mutations in the germline of the mismatch repair gene (eg, the MLH1 gene). If a pathogenic variant is identified, it is a definitive diagnosis of Lynch syndrome.

It has been reported that about 16% of the patients diagnosed with MSI-H had Lynch syndrome, and it is a fact that the MSI test alone cannot sufficiently narrow down Lynch syndrome. The reason for this is that MSI-H cancer is found not only in Lynch syndrome but also in sporadic solid tumors (reported to be found in ~ 15% of sporadic colorectal cancers). Therefore, by distinguishing between Lynch syndrome and sporadic MSI-H cancer, it is possible to narrow down Lynch syndrome more accurately.

The clinical feature of sporadic MSI-H colorectal cancer is that it occurs frequently in elderly women and the right large intestine. As for mismatch repair proteins, Lynch syndrome had many MLH1 and MSH2 deficiencies, whereas most of them had MLH1 protein deficiencies. In 1998, it was reported that methylation of CpG islands in the promoter region of the MLH1 gene, which encodes the MLH1 protein, is responsible for the MLH1 protein deficiency. That is, in cancers with MLH1 deficiency, sporadic MSI-H colorectal cancer and Lynch syndrome can be distinguished by the presence or absence of methylation of CpG islands in the MLH1 gene.

For example, Patent Document 1 describes a method for detecting the methylation of CpG islands in the MLH1 gene by a methylation-sensitive enzyme, and simultaneously amplifying the CpG islands in the MLH1 gene and the region containing V600E in the BRAF gene to simultaneously amplify the CpG islands. A method for detecting methylation by a methylation-sensitive enzyme and V600E in the BRAF gene by sequence analysis is disclosed.

Here, the V600E mutation in the BRAF gene is found in hairy cell leukemia, malignant melanoma, thyroid cancer, ovarian cancer, colorectal cancer, etc., and about 10% in colorectal cancer. Although 35 to 43% of sporadic colorectal cancers showing MSI-H have V600E mutations in the BRAF gene in tumor tissue, most colorectal cancers in Lynch syndrome show MSI-H but V600E in the BRAF gene. Almost no mutations are found. Therefore, the presence or absence of the V600E mutation in the BRAF gene may be used to distinguish between sporadic colorectal cancer and Lynch syndrome.

Patent Document 1 discloses a specific region for detecting methylation of the MLH1 gene, and when methylation cytosine in the region is detected by a methylation sensitivity restriction enzyme cleavage method and methylation is detected. Discloses that the subject is determined not to have Lynch syndrome or Lynch-like. Further, Patent Document 1 discloses that a predetermined region of the BRAF gene is amplified at the same time as the above-mentioned methylation of the MLH1 gene, and the presence or absence of a V600E mutation in the BRAF gene is investigated.

Further, Patent Document 2 discloses a cancer-specific variant for detecting a mutation (V600E) in the BRAF gene, a method for diagnosing cancer, and a method for developing a therapeutic agent for treating cancer. ..

Japanese Unexamined Patent Publication No. 2019-135928 Japanese Patent No. 4643909

As described above, although it is known to detect a mutation (V600E) in a specific region for detecting methylation of the MLH1 gene or a BRAF gene, a technique for simultaneously detecting these with high accuracy is known. I didn't. Therefore, in view of such circumstances, it is an object of the present invention to provide a kit capable of detecting methylation of the MLH1 gene and mutation (V600E) in the BRAF gene with high accuracy.

As a result of diligent studies to solve the above problems, the present inventors have obtained a probe for detecting a region encoding a mutation (V600E) in the BRAF gene based on a sequence in which cytosine in the region is replaced with thymine. By designing, we found that methylation of the MLH1 gene and mutation (V600E) in the BRAF gene can be detected simultaneously with high accuracy, and the present invention was completed.

The present invention includes the following.
(1) A step of treating genomic DNA prepared from a sample with hydrogen sulfite, a region for detecting methylation of the MLH1 gene using genomic DNA treated with hydrogen sulfite as a template, and a V600E mutation site in the BRAF gene. The step of amplifying the region containing the A method for simultaneously detecting methylation of the MLH1 gene and V600E mutation of the BRAF gene in a sample, including the steps of
(2) Regarding the region for detecting methylation of the MLH1 gene, a methylation-compatible probe corresponding to the amplicon when the region is methylated and a demethylation corresponding to the amplicon when the region is not methylated. The method according to (1), wherein a probe set including a corresponding probe is used.
(3) The method according to (2), wherein the methylation-compatible probe contains the base sequence of SEQ ID NO: 17, and the non-methylation-compatible probe contains the base sequence of SEQ ID NO: 16.
(4) Regarding the region containing the V600E mutation site in the BRAF gene, a wild-type probe corresponding to the case where the V600E mutation site is a wild type and a mutant probe corresponding to the case where the V600E mutation site is a mutant type. The method according to (1), wherein a probe set consisting of the above is used.
(5) The method according to (4), wherein the wild-type probe contains the base sequence of SEQ ID NO: 13, and the mutant probe contains the base sequence of SEQ ID NO: 14.
(6) It is characterized by comprising a step of mixing a blocking nucleic acid having a base sequence that hybridizes to an amplicon having a wild-type V600E mutation site with a solution containing an amplicon obtained by amplification (1). The method described.
(7) The method according to (6), wherein the blocking nucleic acid contains the base sequence of SEQ ID NO: 15.
(8) The method according to (1), wherein the microarray in which the probe is fixed to a substrate is used to detect the amplicon.

(9) A region for detecting methylation of the MLH1 gene and containing a probe for the MLH1 gene corresponding to the base sequence of the region after hydrogen sulfite treatment and a V600E mutation site in the BRAF gene. A kit that simultaneously detects methylation of the MLH1 gene and V600E mutation of the BRAF gene in a sample, including a probe set, including a probe for the BRAF gene corresponding to the base sequence of the region after hydrogen sulfite treatment.
(10) The probe for the MLH1 gene includes a methylation-compatible probe corresponding to an amplicon when the region is methylated and a non-methylation-compatible probe corresponding to an amplicon when the region is not methylated. The kit according to (9).
(11) The kit according to (10), wherein the methylation-compatible probe contains the base sequence of SEQ ID NO: 17, and the non-methylation-compatible probe contains the base sequence of SEQ ID NO: 16.
(12) The BRAF gene probe is characterized by including a wild-type probe corresponding to the case where the V600E mutation site is a wild type and a mutant probe corresponding to the case where the V600E mutation site is a mutant type. The kit described in (9).
(13) The kit according to (12), wherein the wild-type probe contains the base sequence of SEQ ID NO: 13, and the mutant probe contains the base sequence of SEQ ID NO: 14.
(14) The kit according to (9), wherein the V600E mutation site contains a blocking nucleic acid having a base sequence that hybridizes to a wild-type amplicon.
(15) The kit according to (14), wherein the blocking nucleic acid contains the base sequence of SEQ ID NO: 15.
(16) The kit according to (9), wherein the probe for the MLH1 gene and the probe for the BRAF gene include a microarray immobilized on a substrate.

According to the present invention, since the probe for detecting the region containing the V600E mutation site in the BRAF gene is designed based on the base sequence after bisulfite treatment, the methylation of the MLH1 gene and the sudden V600E in the BRAF gene are designed. Mutations can be detected simultaneously with high accuracy. Therefore, by applying the present invention, it is possible to quickly and efficiently distinguish between sporadic colorectal cancer and Lynch syndrome.

It is a figure which shows the base sequence of a part of the MLH1 gene containing the base which was the detection target in this Example, and part of the BRAF gene. It is a figure which shows the base sequence after the part of the MLH1 gene and part of the BRAF gene shown in FIG. 1 were treated with bisulfite (bisulfite treatment). It is a characteristic diagram which shows the result of having measured the fluorescence intensity of BRAF / MLH1 under the conditions 1, 2 and 3 of the hybridaization experiment performed in this Example. It is a characteristic diagram showing the result of calculating the judgment value (= strength of mutant probe / (strength of wild-type probe + strength of mutant probe)) for four types of controls having different methylation frequencies performed in the examples. A shows the result of determining the methylation of the MLH1 gene, and B shows the result of determining the V600E mutation in the BRAF gene. It is a characteristic diagram which shows the result of having performed PCR by applying the PCR program shown in Table 4 or the PCR program shown in Table 9, and measuring the fluorescence intensity from each probe.

Hereinafter, the present invention will be described in detail.

<Methylation of MLH1 gene>
The MLH1 gene (MutL homolog 1 gene) is a gene located on chromosome 3 and encoding a type of mismatch repair protein. By methylation of the MLH1 gene, the expression of the MLH1 gene is suppressed. Methylation of the MLH1 gene can result in cancers such as colon cancer, small bowel cancer, uterine body cancer, ovarian cancer, gastric cancer, renal pelvis / urinary tract cancer, pancreatic cancer, biliary tract cancer, brain tumor, Muir-Torre syndrome, malignant melanoma, non-malignant melanoma. It is also found in other cancers such as small cell lung cancer, prostate cancer, and thyroid cancer.

Methylation that suppresses the expression of the MLH1 gene is determined by hydrogen sulfite treatment (also referred to as bisulfite treatment), which will be described in detail later. In particular, for methylation that suppresses the expression of the MLH1 gene, it is preferable to detect the methylation of cytosine in the CpG sequence contained in the CpG island that appears in the MLH1 gene (including the expression control region and the coding region). CpG islands are defined as regions where CG sequences (dinucleotides consisting of 5'-CG-3') frequently appear in specific regions. Here, assuming that there is no bias in the types of bases (A, G, C and T) that appear in a predetermined genomic region, the probability of appearance of a dinucleotide consisting of 5'-CG-3'is 1/16. Therefore, the region in which the dinucleotide consisting of 5'-CG-3'appears frequently is the region in which the appearance probability of the dinucleotide consisting of 5'-CG-3'appearing in the region exceeds 1/16. Can be defined. Further, the region in which the dinucleotide consisting of 5'-CG-3'appears frequently is not limited to the above definition, and the GC content contained in the region exceeds 50% and is present in 5'. -The CG-3'ratio may be defined as 60% or more (CpG observed / expected> 0.6) of the amount expected from the GC content.

More specifically, as methylation that suppresses the expression of the MLH1 gene, methylation in the region disclosed in Herman JG. Et al., Proc. Natl. Acad. Sci., 95, 6870-6875, 1998 was discriminated. Alternatively, the methylation of the CpG sequence in the marker region for determining the MLH1 methylation group disclosed in JP-A-2019-135928 may be discriminated. The base sequence of the marker region for determining the MLH1 methylation group disclosed in JP-A-2019-135928 is shown in SEQ ID NO: 1.

For methylation that suppresses the expression of the MLH1 gene, it is particularly preferable to detect the methylation of cytosine in the CpG sequence contained in the nucleotide sequence shown in SEQ ID NO: 1. The cytosine to be detected is not particularly limited as long as it is cytosine in the CpG sequence of the base sequence shown in SEQ ID NO: 1. The positions of cytosine that detect methylation include, for example, the 1st, 7th, 38th, 47th, 61st, 78th, 85th, 95th, 97th, 103rd, and 122nd positions of the base sequence of SEQ ID NO: 1. , 133rd, 148th, 217th, 240th, 264th, 272th, 305th, 320th, 340th, 351st, 355th, 371th, 374th, 382th, 398th, 403th, 405th Rank, 408, 425, 466, 476, 481, 500, 508, 513, 515, 528, 530, 536, 548, 567, 580, 583, Cytosine at 590th, 593th, 605th, 631st, 638th, 653rd, and 663rd.

The cytosine for detecting methylation may be one of these cytosines, two cytosines, 3 or more, 4 or more, 5 or more, 10 or more cytosines, or all cytosines. May be. Specifically, the position of the cytosine that detects methylation can be preferably the cytosine at position 264 or the cytosine at position 272 in SEQ ID NO: 1, and more preferably the cytosine at position 264 and position 272 in SEQ ID NO: 1. Can be cytosine.

<V600E mutation in BRAF gene>
The BRAF gene is a gene encoding a BRAF (v-raf murine sarcoma viral oncogene homolog B1) protein, and BRAF activated by a mutation in which valine at position 600 becomes glutamic acid (V600E mutation) activates the MAPK pathway. It is known to mutate and excite abnormal cell proliferation. The V600E mutation in the BRAF gene is a gene mutation found in lung cancer, colon cancer, thyroid cancer, biliary tract cancer and the like. The base sequence of a part of the BRAF gene containing the V600E gene mutation is shown in SEQ ID NO: 2. In the V600E gene mutation, A is the first codon (ATG) (not shown in SEQ ID NO: 2) and T at the 1799th position is A, so that the 600th valine counted from the N-terminal of the BRAF protein is It will mutate to glutamic acid. In SEQ ID NO: 2, w (T or A) at position 267 is the position of the V600E gene mutation.

<Methylation of MLH1 gene and detection of V600E mutation in BRAF gene>
To detect the methylation of the MLH1 gene and the V600E mutation of the BRAF gene described above, first, the genomic DNA prepared from the sample is treated with bisulfite. Here, the sample means a biological sample obtained from a test subject such as a patient suspected of having a disease. The biological sample is not particularly limited, but is limited to sheep water containing fetal or embryo-derived cells, fetal-derived cell samples such as navel cord, body fluid samples such as blood, ascites, and saliva, cancer tissues or tissues suspected of having cancer, and oral mucosal cells. , Hair, skin can be mentioned. The method for extracting genomic DNA is not particularly limited, and a conventionally known method or a method using a commercially available genomic DNA extraction reagent kit can be appropriately used.

Bisulfite treatment is also called bisulfite treatment, and unmethylated cytosine residues contained in genomic DNA are converted to uracil. On the other hand, methylated cytosine residues contained in genomic DNA are not converted. For example, in the CpG island contained in genomic DNA, if the cytosine of the 5'-CG-3'dinucleotide is not methylated, the dinucleotide is converted to 5'-UG-3'. Also, if cytosine, a dinucleotide consisting of 5'-CG-3', is methylated, the dinucleotide remains 5'-CG-3'.

Here, the concentration of bisulfite added during treatment with bisulfite is not particularly limited as long as it can sufficiently convert unmethylated cytosine in genomic DNA, but is not particularly limited, for example, in a solution containing genomic DNA. The final concentration is 1 M or more, preferably 1 to 15 M, and more preferably 3 to 10 M. The incubation conditions (temperature and time) after the addition of bisulfite can be appropriately set according to the amount of bisulfite added. For example, when bisulfite is added at a final concentration of 6 M, 50 to Incubate at 80 ° C for 10 minutes to 7 hours.

Next, using the genomic DNA treated with bisulfite as a template, the region for detecting methylation of the MLH1 gene and the region containing the V600E mutation site in the BRAF gene are each amplified by a pair of primers.

The base length of the region to be amplified is not particularly limited, but can be several tens to several thousand bases, particularly preferably 50 to 1000 bases, and 50 to 700 bases. It is more preferably 50 to 500 bases long, further preferably 50 to 300 bases, and even more preferably 50 to 100 bases. The region for detecting methylation of the MLH1 gene and the region containing the V600E mutation site in the BRAF gene are not particularly limited, but are preferably substantially the same base length. The region for detecting methylation of the MLH1 gene and the region containing the V600E mutation site in the BRAF gene are preferably the same base length, but may have different base lengths. When the base lengths of the region for detecting methylation of the MLH1 gene and the region containing the V600E mutation site in the BRAF gene are different, the different base length is preferably, for example, 20 bases or less, preferably 15 bases or less. It is more preferably 10 bases or less, and most preferably 5 bases or less.

The region for detecting methylation of the MLH1 gene is not particularly limited, but may be, for example, a region consisting of the above-mentioned nucleotide sequence of SEQ ID NO: 1 or a part of the region contained in the nucleotide sequence of SEQ ID NO: 1. As described above, the base sequence of SEQ ID NO: 1 includes the 1st, 7th, 38th, 47th, 61st, 78th, 85th, 95th, 97th, 103rd, 122nd, and 133rd positions. 148th, 217th, 240th, 264th, 272th, 305th, 320th, 340th, 351st, 355th, 371th, 374th, 382th, 398th, 403th, 405th, 408th , 425th, 466th, 476th, 481st, 500th, 508th, 513th, 515th, 528th, 530th, 536th, 548th, 567th, 580th, 583th, 590th, 593th Cytosine, which constitutes CpG, exists at positions, 605, 631, 638, 653, and 663. A region containing one or more cytosines selected from these can be a region for detecting methylation of the MLH1 gene. More specifically, when detecting the methylation of cytosine at position 264 and cytosine at position 272 in SEQ ID NO: 1, in order to detect the methylation of the MLH1 gene in the region containing these cytosine at position 264 and cytosine at position 272. It can be an area.

A pair of primers for amplifying the region for detecting methylation of the MLH1 gene can be appropriately designed according to a conventional method. For example, when the region for detecting methylation of the MLH1 gene is a predetermined partial region in SEQ ID NO: 1, the pair of primers can be designed based on the base sequence of SEQ ID NO: 1. For example, when the region containing cytosine at position 264 and cytosine at position 272 is used as a region for detecting methylation of the MLH1 gene, the forward primer is 5'more than cytosine at position 264 in the base sequence of SEQ ID NO: 1. It can be designed at a position separated by several bases in the side (upstream) direction, for example, 1 to 5 bases, 1 to 10 bases, 1 to 20 bases, and 1 to 30 bases. Similarly, as a reverse primer, several bases, for example, 1 to 5 bases, 1 to 10 bases, 1 to 20 bases, 1 to 1 to 3'side (downstream) from cytosine at position 272 in the base sequence of SEQ ID NO: 1. It can be designed at a position 30 bases away.

At this time, as a pair of primers for amplifying the region for detecting methylation of the MLH1 gene, hybridize to a region containing no dinucleotide consisting of 5'-CG-3'constituting CpG islands. It is preferable to design in. This is because the region for detecting methylation of the MLH1 gene is amplified using the genomic DNA after hydrogen sulfite treatment as a template. That is, since cytosine other than the dinucleotide consisting of 5'-CG-3'is not methylated, it is converted to uracil by hydrogen sulfite treatment. Therefore, using the genomic DNA after treatment with hydrogen sulfite as a template, a pair of primers for amplifying the region for detecting methylation of the MLH1 gene is from 5'-CG-3'in the base sequence of SEQ ID NO: 1. It will be designed based on the base sequence in which cytosine other than the dinucleotide is replaced with uracil (or thymine).

On the other hand, the region containing the V600E mutation site in the BRAF gene is not particularly limited, but may be, for example, a region consisting of the above-mentioned nucleotide sequence of SEQ ID NO: 2 or a partial region contained in the nucleotide sequence of SEQ ID NO: 2. .. The V600E mutation site is a mutation in which A is the first (not shown in SEQ ID NO: 2) of the start codon (ATG) and T at the 1799th position is A. Therefore, the region containing the V600E mutation site in the BRAF gene can be rephrased as the region containing the 1799th base with A as the first codon (ATG). In addition, based on the base sequence of SEQ ID NO: 2, the region containing the V600E mutation site in the BRAF gene can be paraphrased as the region containing the 267th base in SEQ ID NO: 2.

A pair of primers for amplifying the region containing the V600E mutation site in the BRAF gene can also be appropriately designed according to a conventional method. For example, a pair of primers for amplifying the region containing the V600E mutation site in the BRAF gene can be designed based on the base sequence of SEQ ID NO: 2. That is, the pair of primers is designed as a forward primer and a reverse primer so as to amplify the region of the start codon (ATG) containing the 1799th base (267th base in SEQ ID NO: 2) with A as the first. .. For example, as a forward primer, several bases, for example, 1 to 5 bases, 1 to 10 bases, 1 to 20 bases, or 1 to 30 bases in the 5'side (upstream) direction from the 267th base in the base sequence of SEQ ID NO: 2. It can be designed at a position separated from the base. Similarly, as the reverse primer, several bases, for example, 1 to 5 bases, 1 to 10 bases, 1 to 20 bases, or 1 to 1 to 3'side (downstream) from the 267th base in the base sequence of SEQ ID NO: 2. It can be designed at a position 30 bases away.

Here, the region containing the V600E mutation site in the BRAF gene is also amplified using the genomic DNA after hydrogen sulfite treatment as a template. Since cytosine contained in the BRAF gene present in genomic DNA is not methylated, it is converted to uracil by bisulfite treatment. Therefore, using the genomic DNA after hydrogen sulfite treatment as a template, the pair of primers for amplifying the region containing the V600E mutation site in the BRAF gene replaces cytosine in the nucleotide sequence of SEQ ID NO: 2 with uracil (or thymine). It will be designed based on the base sequence.

At this time, as the amplification reaction, a polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, etc. can be applied. In the amplification reaction, it is desirable to add a label so that the amplified amplicon can be identified. At this time, the method for labeling the amplified amplicon is not particularly limited, but for example, a method in which a primer used for the amplification reaction is labeled in advance may be used, or a labeled nucleotide may be used as a substrate for the amplification reaction. You may use the method you use. The labeling substance is not particularly limited, but a radioisotope, a fluorescent dye, or an organic compound such as digoxigenin (DIG) or biotin can be used.

In addition, this reaction system includes a buffer required for nucleic acid amplification / labeling, a heat-resistant DNA polymerase, a primer specific to the amplification region, and a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate to which a fluorescent label or the like is added). , Nucleotide triphosphate, magnesium chloride and the like.

Next, as described above, the region for detecting the methylation of the amplified MLH1 gene and the region containing the V600E mutation site in the BRAF gene are detected using the probe for the MLH1 gene and the probe for the BRAF gene, respectively.

The regions for detecting methylation of the MLH1 gene include a methylation-compatible probe corresponding to the case where the methylation site is methylated and a non-methylation-compatible probe corresponding to the case where the methylation site is not methylated. It can be detected using a probe for the MLH1 gene that contains it. That is, the methylation-compatible probe corresponds to the case where the cytosine of the dinucleotide composed of 5'-CG-3'is methylated, and the cytosine of 5'-CG-3'is maintained even by the above-mentioned bisulfite treatment. Therefore, it has a sequence corresponding to the dinucleotide consisting of 5'-CG-3'. On the other hand, the non-methylation-compatible probe corresponds to the case where the dinucleotide consisting of 5'-CG-3'is not methylated, and the cytosine of 5'-CG-3'is converted to uracil by the above-mentioned hydrogen sulfite treatment. It will have a sequence corresponding to the dinucleotide consisting of 5'-UG-3'(5'-TG-3' as an amplicon). Hereinafter, a pair of probes consisting of a methylation-compatible probe and a non-methylation-compatible probe may be simply referred to as a probe set or a pair of probe sets. These probe sets preferably have the same sequence except for the methylation site to be detected (cytosine of the dinucleotide consisting of 5'-CG-3').

In particular, if the region for detecting methylation of the MLH1 gene contains multiple methylation sites (5'-CG-3'dinucleotides), methylation of one or more of these multiple methylation sites. Methylation in the partial region containing the site can be detected with a pair of probe sets. At this time, the pair of probe sets may have a sequence corresponding to one methylation site contained in the region for detecting methylation of the MLH1 gene, or may correspond to two or more methylation sites. It may have a sequence. More specifically, a pair of probe sets can have sequences corresponding to 2-7 methylation sites and can also have sequences corresponding to 2-4 methylation sites. Probe specificity can be enhanced by using probe sets that correspond to multiple methylation sites.

In addition, a pair of probe sets that detect methylation can be designed for each of multiple methylation sites contained in the region for detecting methylation of the MLH1 gene. With these multiple probe sets, multiple methylation sites can also be detected independently. By preparing a plurality of probe sets, all the methylation sites contained in the amplicon can be targeted for detection, but some methylation sites can be selected and targeted for detection.

On the other hand, the region containing the V600E mutation site in the BRAF gene includes a wild-type probe corresponding to the case where the V600E mutation site is wild-type and a mutant probe corresponding to the case where the site is mutant type. It can be detected using a probe for. That is, the wild-type probe is designed to hybridize to the base sequence in which the 1799th base (267th base in SEQ ID NO: 2) is T among the start codons (ATG) in the BRAF gene, with A as the first. To. In addition, the mutant probe is designed to hybridize to the base sequence in which the 1799th base (267th base in SEQ ID NO: 2) is A among the start codons (ATG) in the BRAF gene. To.

Here, the region containing the V600E mutation site in the BRAF gene is amplified using the genomic DNA after hydrogen sulfite treatment as a template, as described above. Since cytosine contained in the BRAF gene present in genomic DNA is not methylated, it is converted to uracil by bisulfite treatment. Therefore, in the region containing the V600E mutation site in the BRAF gene, which was amplified using the genomic DNA after bisulfite treatment as a template, the position originally cytosine is amplified as thymine. Therefore, the wild-type probe and the mutant probe are designed based on the base sequence in which cytosine in SEQ ID NO: 2 is replaced with thymine, respectively.

The base length of these probes is not particularly limited, but can be, for example, 10 to 30 base lengths, preferably 15 to 25 base lengths. Also, the probe designed as described above is preferably nucleic acid, more preferably DNA. The DNA includes both double-stranded and single-stranded DNA, but is preferably single-stranded DNA. The probe can be obtained, for example, by chemically synthesizing it with a nucleic acid synthesizer. As the nucleic acid synthesizer, a device called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer, or the like can be used.

The probe designed as described above is preferably used in the form of a microarray (as an example, a DNA chip) by immobilizing its 5'end on a carrier. At this time, the microarray is provided with a probe set for detecting methylation or unmethylation at a plurality of methylation sites contained in the above-mentioned amplicon. Further, a plurality of probe sets corresponding to different partial regions may be provided.

As the material of the carrier, those known in the art can be used and are not particularly limited. For example, precious metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductive materials such as carbon represented by graphite and carbon fiber; single crystal silicon, amorphous. Silicon materials typified by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials typified by SOI (silicon on insulator); glass, quartz glass, alumina, sapphire, ceramics, fol Inorganic materials such as sterite and photosensitive glass; polyethylene, ethylene, polyprovylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluororesin, polyvinyl chloride, polyvinylidene chloride, polyvinylacetate, polyvinyl alcohol, polyvinyl acetal , Acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and organic such as polysulfone. Materials and the like can be mentioned. The shape of the carrier is also not particularly limited, but is preferably flat.

In particular, as the carrier, it is preferable to use a carrier having a carbon layer and a chemically modifying group on the surface. The carrier having a carbon layer and a chemical modification group on the surface includes a carrier having a carbon layer and a chemical modification group on the surface of the substrate and a carrier having a chemical modification group on the surface of the substrate composed of the carbon layer. As the material of the substrate, those known in the art can be used, and the same materials as those mentioned above as the carrier materials can be used without particular limitation.

In a microarray, a carrier having a fine flat plate-like structure is preferably used. The shape is not limited to rectangular, square and round, but usually 1 to 75 mm square, preferably 1 to 10 mm square, and more preferably 3 to 5 mm square are used. Since it is easy to produce a carrier having a fine flat plate-like structure, it is preferable to use a substrate made of a silicon material or a resin material, and in particular, a carrier having a carbon layer and a chemically modifying group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon contains a slight change in the orientation of the crystal axis in a partial part (sometimes called a mosaic crystal) or an atomic scale disorder (lattice defect). Is also included.

The carbon layer formed on the substrate is not particularly limited, but is limited to synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, and carbon-based material (for example, graphite, fullerene, carbon nanotube). ), A mixture thereof, or a laminate thereof is preferable. Further, 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 and the like may be used. Here, soft diamond is a general term for incomplete diamond structures that are a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC: Diamond Like Carbon), and the mixing ratio thereof is not particularly limited. The carbon layer has excellent chemical stability and can withstand subsequent reactions in the introduction of chemically modifying groups and the bond with the substance to be analyzed, and the bond is flexible because it is bonded to the substance to be analyzed by electrostatic bonding. It is advantageous in that it has the property, it is transparent to the detection system UV because it does not absorb UV, and it can be energized during electroblotting. It is also advantageous in that there is little non-specific adsorption in the binding reaction with the substance to be analyzed. As described above, a carrier whose substrate itself is made of a carbon layer may be used.

The carbon layer can be formed by a known method. For example, microwave plasma CVD (Chemical vapor deposit) method, ECRCVD (Electric cyclotron resonance chemical vapor deposit) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) sputtering method, ionized vapor deposition method, arc. Examples include a formula vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

In the high frequency plasma CVD method, the raw material gas (methane) is decomposed by the glow discharge generated between the electrodes by the high frequency, and a carbon layer is synthesized on the substrate. In the ionization vapor deposition method, thermions generated by the tungsten filament are used to decompose and ionize the raw material gas (benzene), and a carbon layer is formed on the substrate by the bias voltage. A carbon layer may be formed by an ionization vapor deposition method in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of remaining methane gas.

In the arc-type vapor deposition method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to generate carbon atom plasma from the cathode and evaporate source. By applying a more negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

In the laser vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light to melt it and depositing carbon atoms on a glass substrate.

When a carbon layer is formed on the surface of a substrate, the thickness of the carbon layer is usually about a monolayer to 100 μm, and if it is too thin, the surface of the underlying substrate may be locally exposed, and conversely, it is thick. In this case, the productivity is deteriorated, so it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

By introducing a chemically modifying group on the surface of the substrate on which the carbon layer is formed, the oligonucleotide probe can be firmly immobilized on the carrier. The chemically modifying group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group and an active ester group.

The introduction of the amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet rays in ammonia gas or by plasma treatment. Alternatively, it can be carried out by irradiating the carbon layer with ultraviolet rays in chlorine gas to chlorinate it, and then irradiating it with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting a polyhydric amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.

The introduction of the carboxyl group can be carried out, for example, by reacting the amino acidized carbon layer as described above with an appropriate compound. Examples of the compound used for introducing a carboxyl group are represented by the formula: X-R1-COOH (in the formula, X represents a halogen atom and R1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms). Halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; Among them, R2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid. , Polycarboxylic acids such as polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; formula: R3-CO-R4-COOH (in the formula, R3 is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms. , R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms); ketoic acid or aldehyde acid; formula: X-OC-R5-COOH (in the formula, X is a halogen atom, R5 is a single bond or Represents a divalent hydrocarbon group having 1 to 12 carbon atoms.) Monohalides of dicarboxylic acids, such as succinic acid monoclonal acid, malonic acid monoclonal acid; phthalic acid anhydride, succinic acid anhydride, oxalic acid anhydride, maleine anhydride. Examples thereof include acid anhydrides and acid anhydrides such as butanetetracarboxylic acid anhydride.

The introduction of the epoxy group can be carried out, for example, by reacting the amino acidized carbon layer as described above with an appropriate polyvalent epoxy compound. Alternatively, it can be obtained by reacting an organic peracid with a carbon-carbon double bond contained in the carbon layer. Examples of the organic peracetic acid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, trifluoroperacetic acid and the like.

The introduction of the formyl group can be carried out, for example, by reacting the amino acidized carbon layer as described above with glutaraldehyde.

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

The active ester group means an ester group that has an electron-attracting group with high acidity on the alcohol side of the ester group and activates the nucleophilic reaction, that is, an ester group with high reaction activity. It is an ester group that has an electron-withdrawing group on the alcohol side of the ester group and is more activated than the alkyl ester. The active ester group has reactivity with a group such as an amino group, a thiol group and a hydroxyl group. More specifically, phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. are active ester groups having much higher activity than alkyl esters and the like. Known as. More specifically, examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalateimide group, 5-norbornen-2,3-dicarboxyimide group and the like. In particular, an N-hydroxysuccinimide group is preferably used.

To introduce the active ester group, for example, the carboxyl group introduced as described above is combined with a dehydration condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N-. It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, a group to which an active ester group such as an N-hydroxysuccinimide group is bonded can be formed at the end of the hydrocarbon group via an amide bond (Japanese Patent Laid-Open No. 2001-139532).

The probe is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted onto a carrier by a spotter device or the like. Can produce microarrays immobilized on a carrier. Alternatively, the spotting solution may be manually spotted with a micropipettor.

After spotting, it is preferable to carry out incubation because the reaction in which the probe binds to the carrier proceeds. Incubation is usually carried out at a temperature of −20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is preferably performed in a high humidity atmosphere, for example, in a humidity of 50 to 90%. Following the incubation, it is preferable to perform washing with a washing solution (for example, 50 mM TBS / 0.05% Tween20, 2 × SSC / 0.2% SDS solution, ultrapure water, etc.) in order to remove DNA that is not bound to the carrier. ..

Hybridization reaction is performed between the amplicon obtained as described above and a probe immobilized on a carrier (methylation-compatible probe and non-methylation-compatible probe, and wild-type probe and mutant probe). Methylation of the MLH1 gene can be detected based on hybrids to methylated and non-methylated probes, and V600E mutations in the BRAF gene can be detected based on hybrids to wild-type and mutant probes. be able to. At this time, the solution containing the amplicon preferably contains a blocking nucleic acid that hybridizes to a base sequence (that is, wild type) that does not have a V600E mutation in the BRAF gene. The blocking nucleic acid has a base sequence complementary to a nucleic acid fragment having a wild-type sequence in the region containing the V600E mutation site in the BRAF gene amplified by a pair of primers. By using a blocking nucleic acid, it is possible to detect a region containing a V600E mutation site in the BRAF gene having a mutant sequence with higher accuracy.

In particular, in the above-mentioned method, the genomic DNA after treatment with bisulfite is used as a template to amplify the region containing the V600E mutation in the BRAF gene, and the amplified region contains a large amount of adenine and thymine (AT). rich). At this time, by making the base length of the blocking nucleic acid corresponding to the wild type shorter than that of the wild type probe, even if the amplification product is AT-rich, the wild type amplification product and the blocking nucleic acid are specifically hybridized. It facilitates the formation of soybeans and can be detected with high accuracy even when the above-mentioned region having a variant is contained only in a trace amount.

The hybridization between the probe fixed on the carrier and the amplicon can be detected based on the signal from the label attached to the amplicon. For the signal from the label, for example, when a fluorescent label is used, the fluorescent signal is detected by using a fluorescent scanner, and the signal intensity can be quantified by analyzing this with image analysis software. The hybridization reaction is preferably carried out under stringent conditions. The stringent condition is a condition in which a specific hybrid is formed and a non-specific hybrid is not formed. For example, after a hybridization reaction at 50 ° C. for 16 hours, 2 × SSC / 0.2% SDS, 25 Refers to the conditions for cleaning at ℃, 10 minutes and 2 × SSC, 25 ℃, 5 minutes. Alternatively, the hybridization temperature can be 45 to 60 ° C. when the salt concentration is 0.5 × SSC, and it is more preferable to lower the hybridization temperature when the chain length of the probe is short. When the chain length is long, it is more preferable to set the hybridization temperature higher than this. Needless to say, the higher the salt concentration, the higher the hybridization temperature having specificity, and conversely, the lower the salt concentration, the lower the hybridization temperature having specificity.

In addition, a probe set corresponding to a partial region containing one or more methylation sites among a plurality of methylation sites included in the above-mentioned region for detecting methylation of the MLH1 gene (methylation-compatible probe and non-methylation-compatible probe). When microarrays with probes) are used, the signal intensities from these probe sets can be used to determine methylation or demethylation for the methylation site. Specifically, for a pair of probe sets, the signal intensity in the methylation-compatible probe and the signal intensity in the non-methylation-compatible probe are measured, respectively, and a judgment value for evaluating the signal intensity derived from the methylation-compatible probe is calculated. do. As an example of calculating the judgment value, for example, a method using the formula: [Signal intensity derived from methylation-compatible probe] / ([Signal intensity derived from methylation-compatible probe] + [Signal intensity derived from non-methylation-compatible probe]). Can be mentioned. In this determination value, the theoretical value when a fully methylated sample is measured is 1, and the theoretical value when a completely unmethylated (fully unmethylated) sample is measured is 0.

Then, the judgment value calculated by the above formula is compared with a predetermined threshold value (cutoff value), and if the judgment value exceeds the upper limit threshold value, the region for detecting methylation of the MLH1 gene is used. It is determined that the included CpG island is methylated, and if the determination value is below the lower limit threshold value, it is determined that the included CpG island is not methylated. If the determination value is between the upper and lower thresholds set in advance, it can be determined that about half of the CpG islands contained in the region are methylated (one allele is methylated). can. A single threshold value may be set, and if it exceeds this threshold value, it may be determined that it is methylated, and if it is lower than this threshold value, it may be determined that it is not methylated. By using the determination value in this way, it is possible to determine the methylation of the CpG island contained in the region for detecting the methylation of the MLH1 gene described above.

As described above, the V600E mutation in the BRAF gene can be detected using the genomic DNA after bisulfite treatment (bisulfite treatment) for detecting the methylation of the MLH1 gene. That is, according to the method described above, methylation of the MLH1 gene and V600E mutation in the BRAF gene can be detected at the same time. By detecting the methylation of the MLH1 gene and the V600E mutation in the BRAF gene, for example, Lynch syndrome and sporadic colorectal cancer in colorectal cancer can be discriminated.

Specifically, in colorectal cancer, if the MLH1 gene is methylated and the BRAF gene has a V600E mutation, it can be determined to be sporadic MSI-H colorectal cancer. If there is no methylation of the MLH1 gene and there is a V600E mutation in the BRAF gene, it can be determined that there is a high probability of sporadic colorectal cancer that is not MSI-H. Sporadic MSI-H colorectal cancer can also be determined if the MLH1 gene is methylated and there is no V600E mutation in the BRAF gene. Then, in MSI-H colorectal cancer, if there is no methylation of the MLH1 gene and no V600E mutation in the BRAF gene, it can be judged that there is a high possibility of Lynch syndrome in colorectal cancer.

Hereinafter, the present invention will be described in more detail by way of examples, but the technical scope of the present invention is not limited to the following examples.

[Example 1]
In this example, we developed a method to simultaneously detect methylation of the MLH1 gene and V600E mutation in the BRAF gene, which are used for the diagnosis of Lynch syndrome and sporadic colorectal cancer. FIG. 1 shows the base sequences (SEQ ID NO: 4) of a part of the BRAF gene (SEQ ID NO: 3) and a part of the MLH1 gene (promoter region). Further, FIG. 2 shows the base sequences (SEQ ID NOs: 5 and 6) of the region shown in FIG. 1 after bisulfite treatment (bisulfite treatment). Cytosine becomes uracil by hydrogen sulfite treatment (bisulfite treatment), and in FIG. 2, the uracil is described as thymine for convenience. Further, in FIG. 2, the nucleic acid to be analyzed is surrounded by a square. In FIG. 2, the positions of the primers for amplifying the region containing the nucleic acid to be analyzed are underlined in each base sequence. The base sequence of each primer is shown in Table 1. Table 2 shows the base sequence of the probe that hybridizes with the amplified region.

In order to amplify each region, first, a primer mixture in which the primers shown in Table 1 were mixed at the concentration shown in Experiment 1 described later was prepared.

Next, a PCR reaction solution was prepared so as to have the composition shown in Table 3.

The PCR temperature conditions were set as shown in Table 4.

Then, the hybridization reaction was performed using the MLH1 / BRAF analysis chip (MB chip) prepared in advance. Specifically, first, the hybridization oven was set at 60 ° C., a tapper containing 30 mL of water was installed, and the mixture was left for 1 hour or more. Next, the hybridization buffer (2.25 × SSC, 0.23% SDS) and the PCR product were taken out from the freezer and returned to room temperature. 4 μl of PCR product and 2 μl of hybridization buffer were mixed. After adding 3 μL of the mixed solution to the chip cover, the mixture was set on the chip. The temperature condition in the hybridization reaction was 60 ° C.

Then, a cleaning solution (0.1 × SSC / 0.1% SDS solution) was prepared, the chip after the hybridization reaction was placed in a stainless steel holder, and the inside of the cleaning solution was washed by moving it up and down 10 times, and allowed to stand for 5 minutes. After cleaning, a stainless steel holder holding the chips was placed in 1 × SSC solution. Then, the water was wiped off, a cover film was covered, and the measurement was performed by the fluorescence intensity estimation method using the gene analyzer BIOSHOT (manufactured by Toyo Kohan Co., Ltd.).

[Experiment 1]
In Experiment 1, we investigated the design of primer concentration for detecting BRAF / MLH1. Specifically, the concentrations of the two types of primer sets corresponding to the BRAF / MLH1 region were carried out under conditions 1 to 3. Each condition is shown in Tables 5 to 7, respectively.

In this experiment, the above-mentioned PCR was performed using RKO cell line-derived DNA after bisulfite treatment (bisulfite treatment) as a sample for evaluating the intensity of BRAF / MLH1. The RKO cell line has an MLH1 methylation frequency of about 0% and a BRAF mutation frequency of about 66%.

The fluorescence intensity of BRAF / MLH1 is shown in FIG. 3 under conditions 1, 2 and 3. In the inspection system, the minimum fluorescence intensity is set to 5000 for stable analysis. As shown in FIG. 3, under condition 1, the fluorescence intensity of BRAF was less than 5000, and the analysis condition was not satisfied. On the other hand, under conditions 2 and 3, the fluorescence intensity of BRAF was detected higher than that of condition 1, and under condition 3, the fluorescence intensity of MLH1 and BRAF could be detected to the same extent.

From the results of this experiment, it was clarified that the fluorescence intensity was stably detected under condition 3, that is, the primer set concentration was in the presence of MLH1: BRAF = 1: 3.75.

[Experiment 2]
In Experiment 2, performance evaluation was performed to verify whether mutations and methylation could be detected. For BRAF, a blocking nucleic acid (blocker) having a sequence similar to that of the wild-type probe was used in order to reduce non-specific binding of the wild-type PCR product to the mutation detection probe. The sequences of the probe set and blocker used in the study are shown in Table 8.

In this experiment, EpiScope Methylated HCT116 gDNA (methylation frequency 95%) and EpiScope Unmethylated HCT116 DKO gDNA (methylation frequency 0%) as MLH1 controls, and RKO cell line-derived DNA (mutation frequency 66%) as BRAF controls and human adults. Using genomic DNA derived from normal tissues (mutation frequency 0%), four types of controls were prepared so that the mutation frequency or methylation frequency was 0%, 5%, 10%, and 15%, respectively. The blocker was mixed with the hybridization buffer so as to have a concentration of 500 nM.

The judgment value, which is the intensity ratio, was calculated from the fluorescence intensity of each probe obtained by using the control treated with bisulfite (bisulfite treatment) from the following formula.
Judgment value = strength of mutant probe / (strength of wild-type probe + strength of mutant probe)

The results of the judgment values obtained from each control are shown in FIG. The number of trials is N = 6, and the error bar is S.D. The kit was intended to determine the presence or absence of mutation and methylation, and the control with 0% mutation and methylation frequency and the error bar of the determination value were separated in other cases. From this result, it was confirmed that the probe and blocker set in this experiment can detect methylation of the MLH1 gene and V600E mutation in the BRAF gene at the same time.

[Example 2]
In this example, PCR conditions were investigated to suppress non-specific amplification of the base sequence in which the conversion from cytosine to uracil did not proceed normally by bisulfite treatment (bisulfite treatment). Specifically, a PCR program in which the annealing temperature and time were changed when the primers designed in Example 1 were used was set (Table 9).

In this experiment, a comparative test was carried out between the PCR program shown in Table 4 set in Example 1 and the PCR program shown in Table 9 set in this example. Using genomic DNA derived from normal human tissue (mutation and methylation frequency 0%), treated and untreated samples with bisulfite were prepared, and 20 ng of each sample was added as a template in PCR. The test conditions other than the PCR program were based on Example 1.
The fluorescence intensity obtained from the conditions of each PCR program is shown in FIG. In the case of the PCR program shown in Table 4, about 4,000 fluorescence intensities were detected from the BRAF wild-type probe and the MLH1 methylation probe even in the sample not treated with bisulfite. It is considered that this is because in the PCR program shown in Table 4, in the annealing reaction, the base sequence untreated with bisulfite and the primer were non-specifically bound to generate an amplicon. From this result, in the PCR program shown in Table 4, even if the base sequence in which the conversion from cytosine to uracil did not proceed normally remains, even if the bisulfite treatment did not proceed normally and the conversion from cytosine to uracil did not proceed normally, the amplicon was used. It became clear that there is a concern that erroneous judgment may occur due to the resulting fluorescence intensity.
On the other hand, according to the PCR program of Table 9 set in this example, when the sample not treated with bisulfite was used, the fluorescence intensity was not detected from any of the BRA and MLH1 probes (FIG. 5). ). According to the PCR program in Table 9 set in this example, when the sample subjected to the bisulfite treatment was used, the fluorescence intensity of 10,000 or more was obtained by the wild type probe of BRAF and the unmethylated probe of MLH1 respectively. It was detected, and it was confirmed that the strength was sufficient for the inspection as in the PCR program shown in Table 4.
From the above results, according to the PCR program in Table 9 in which the annealing temperature and time were changed, if the bisulfite treatment (bisulfite treatment) did not proceed normally, an amplicon would not be generated and an erroneous judgment was made. Can be prevented.

Claims (16)

1. A step of treating genomic DNA prepared from a sample with hydrogen sulfite, a region for detecting methylation of the MLH1 gene using genomic DNA treated with hydrogen sulfite as a template, and a region containing the V600E mutation site in the BRAF gene. A step of amplifying each with a pair of primers, and a step of detecting a region for detecting the methylation of the MLH1 gene contained in the amplicon obtained by the amplification and a region containing the V600E mutation site in the BRAF gene with a probe. A method for simultaneously detecting methylation of the MLH1 gene and V600E mutation of the BRAF gene in a sample, including.
2. Regarding the region for detecting methylation of the MLH1 gene, a methylation-compatible probe corresponding to the amplicon when the region is methylated and a non-methylation-compatible probe corresponding to the amplicon when the region is not methylated. The method according to claim 1, wherein a probe set comprising the same probe set is used.
3. The method according to claim 2, wherein the methylation-compatible probe contains the base sequence of SEQ ID NO: 17, and the non-methylation-compatible probe contains the base sequence of SEQ ID NO: 16.
4. For the region containing the V600E mutation site in the BRAF gene, a probe consisting of a wild-type probe corresponding to the case where the V600E mutation site is wild-type and a mutant probe corresponding to the case where the V600E mutation site is mutant type. The method according to claim 1, wherein a set is used.
5. The method according to claim 4, wherein the wild-type probe contains the base sequence of SEQ ID NO: 13, and the mutant probe contains the base sequence of SEQ ID NO: 14.
6. The method according to claim 1, wherein the solution containing the amplicon obtained by amplification is mixed with a blocking nucleic acid having a base sequence that hybridizes to an amplicon having a wild-type V600E mutation site. ..
7. The method according to claim 6, wherein the blocking nucleic acid contains the base sequence of SEQ ID NO: 15.
8. The method according to claim 1, wherein a microarray in which the probe is fixed to a substrate is used to detect the amplicon.
9. A region for detecting methylation of the MLH1 gene, which contains a probe for the MLH1 gene corresponding to the base sequence of the region after treatment with hydrogen sulfite, and a region containing the V600E mutation site in the BRAF gene, which is a hydrogen sulfite. A kit that simultaneously detects methylation of the MLH1 gene and V600E mutation of the BRAF gene in a sample, including a probe set, including a probe for the BRAF gene corresponding to the base sequence of the region after treatment.
10. The MLH1 gene probe is characterized by including a methylation-compatible probe corresponding to an amplicon when the region is methylated and a non-methylation-compatible probe corresponding to an amplicon when the region is not methylated. The kit according to claim 9.
11. The kit according to claim 10, wherein the methylation-compatible probe contains the base sequence of SEQ ID NO: 17, and the non-methylation-compatible probe contains the base sequence of SEQ ID NO: 16.
12. The BRAF gene probe includes a wild-type probe corresponding to the case where the V600E mutation site is a wild type, and a mutant probe corresponding to the case where the V600E mutation site is a mutant type. Item 9. The kit according to item 9.
13. The kit according to claim 12, wherein the wild-type probe contains the base sequence of SEQ ID NO: 13, and the mutant probe contains the base sequence of SEQ ID NO: 14.
14. The kit according to claim 9, wherein the V600E mutation site contains a blocking nucleic acid having a base sequence that hybridizes to a wild-type amplicon.
15. The kit according to claim 14, wherein the blocking nucleic acid contains the base sequence of SEQ ID NO: 15.
16. The kit according to claim 9, wherein the probe for the MLH1 gene and the probe for the BRAF gene include a microarray immobilized on a substrate.

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