WO2024195263A1 - Method for measuring methylation level of cpg of gene - Google Patents

Abstract

The present invention addresses the problem of providing a method for simply measuring the methylation level of CpG in a transcription regulatory region of a gene to be measured, by performing a restriction enzyme treatment in which restriction enzymes are added only in one stage.　The solution is as follows.　Provided is a method for measuring the methylation level of CpG in a transcription regulatory region and/or a region from +1 nucleotide to +1000 nucleotides from a transcription initiation point in a gene to be measured, the method comprising steps (a) to (d): (a) a step for adding (i) DNA containing the gene to be measured, (ii) at least one restriction enzyme selected from HhaI, HpaII, etc., (iii) a restriction enzyme BstUI, and (iv) a restriction enzyme reaction buffer into a reaction vessel followed by sealing; (b) a step, while keeping the reaction vessel sealed, for performing a restriction enzyme treatment at a temperature of 30-50°C for 0.5-16 hours and then at 50-70°C for 0.5-20 hours; (c) a step for amplifying a part or the full length of a predetermined region in the gene to be measured; and (d) a step for measuring the methylation level of CpG in the DNA in the region having been amplified in step (c).

Description

Method for measuring CpG methylation levels of genes

The present invention relates to a method for measuring the CpG methylation level of a gene to be measured.

In recent years, liquid biopsy has been garnering attention as a method for diagnosing cancer. Liquid biopsy is a method for detecting cancer cells or tumor-specific genetic changes suspended in plasma or serum, and is a safer, simpler test method that is less invasive to patients than diagnosis by tissue biopsy.

The inventors have disclosed, as technologies that can be used for liquid biopsy, a method for predicting the presence or absence of a colon tumor by measuring the methylation level of one or more CpGs in the transcriptional regulatory region of Twist homolog 1 (Twist 1) (see Patent Document 1), a method for predicting the presence or absence of a colon tumor by measuring the methylation level of CpGs in the TWIST1, NDRG4, BMP3, or SEPT9 genes in stool or serum (see Patent Document 2), a method for diagnosing cancer by measuring the methylation level of CpGs in the RUNX3 gene (see Patent Document 3), and the fact that a combination of methylated SEPT9 CpGs in serum and AFP is useful for diagnosing hepatocellular carcinoma (see Non-Patent Documents 1 and 2).

It has also been disclosed that CpG methylation of Homeobox A1 (HOXA1) may be used in cancer diagnosis when combined with other cancer markers (see Non-Patent Documents 3 to 5).

In the above Patent Document 1, sodium hydrogen sulfite treatment (bisulfite treatment) is used, which has problems such as a lack of quantitative accuracy when the degree of methylation is low, and the need for a large amount of biological sample.

In addition, in the above Patent Document 2, there are six CpG sequences in the transcriptional regulatory region of Twist 1, and HhaI, HpaII, and BstUI were used as methylation-sensitive restriction enzymes corresponding to the CpGs to cleave each CpG. Here, in the methylation-sensitive restriction enzyme treatment, if all restriction enzymes are used at once, cleavage is incomplete, so the restriction enzyme treatment is performed by adding restriction enzymes in two stages to increase the efficiency of cleavage by the restriction enzymes and improve the accuracy of methylation detection. However, with this method, it is necessary to open the tube lid after the restriction enzyme treatment once and add another restriction enzyme to perform the restriction enzyme treatment again, which causes problems such as evaporation of about 5-20% of the restriction enzyme reaction buffer, changing the composition of the restriction enzyme reaction buffer, and the enzyme reaction takes time, making the DNA more likely to denature. Furthermore, in the above Patent Document 3, as in the above Patent Document 2, methylation-sensitive restriction enzyme treatment is performed by adding restriction enzymes in two stages.

International Publication No. 2010/113529 International Publication No. 2017/043497 JP 2020-14415 A

Kotoh Y, Suehiro Y, Saeki I, et al. Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma. Hepatol Commun 2020; 4: 461-470. Ayano Yamazaki et al., Screening for hepatocellular carcinoma by liquid biopsy using highly sensitive DNA methylation analysis technology: Comparison of diagnostic performance between methylated SST and methylated SEPT9, Yamaguchi Medical Journal, Vol. 70, No. 3, pp. 89-98, 2021 Paco A, de Bessa Garcia SA, Freitas R. Methylation in HOX Clusters and Its Applications in Cancer Therapy. Cells: 2020; 9: 1613. Chalasani N, Ramasubramanian T, Bhattacharya A, et al. A Novel Blood-Based Panel of Methylated DNA and Protein Markers for Detection of Early-Stage Hepatocellular Carcinoma. Clin Gastroenterol Hepatol 2021; 19: 2597-2605. Kisiel J, Dukek B, Kanipakam R, et al. Hepatocellular Carcinoma Detection by Plasma Methylated DNA: Discovery, Phase I Pilot, and Phase II Clinical Validation. Hepatology. 2019; 69: 1180-1192.

The method described in Patent Document 2 performs restriction enzyme treatment by adding restriction enzymes in two stages. Specifically, in the first stage, two types of restriction enzymes are added to a DNA solution and reacted at 37° C. for 16 hours, and then in the second stage, an additional type of restriction enzyme is added and reacted at 60° C. for 16 hours. Here, in order to perform the restriction enzyme treatment in two stages, a task of adding a restriction enzyme in the second stage occurs after the first stage of restriction enzyme treatment. For example, when 96 samples are handled, the task of adding a restriction enzyme in the second stage is as follows, and it takes about 60 minutes to perform all the steps.
(1) Remove the microtube containing the enzyme-treated DNA solution from the heater (2 minutes)
(2) Centrifuge the microtube (6 minutes)
(3) Open the lid of the microtube (2 minutes)
(4) Add the second-stage restriction enzyme (BstUI) (10 seconds x 96 = 960 seconds = 16 minutes)
(5) Pipette about 20 times to thoroughly mix the DNA solution and enzyme (15 seconds x 96 = 1,440 seconds = 24 minutes).
(6) Close the lid of the microtube and seal it (2 minutes)
(7) Centrifuge the microtube (6 minutes)
(8) Place the microtube in the heater (2 minutes)
Therefore, an object of the present invention is to provide a method for easily measuring the methylation level of CpG in the transcriptional regulatory region of a gene to be measured by performing restriction enzyme treatment in only one step of adding a restriction enzyme.

The inventors conducted extensive research to solve the above problems, and discovered that when methylation-sensitive restriction enzymes with different optimum temperatures are used, the methylation level of CpG in the gene to be measured can be measured even when certain methylation-sensitive restriction enzymes are treated simultaneously at a certain reaction temperature and reaction time, and thus completed the present invention.

That is, the present invention is as follows.
[1] (a) placing in a reaction vessel: (i) DNA encoding a gene to be measured;
(ii) at least one restriction enzyme selected from HhaI, HpaII, Hpy99I, SacII, SmaI, NotI, AatII, AccI, Aor13HI, Aor51HI, BspT104I, BssHII, Cfr10I, ClaI, CpoI, Eco52I, HaeII, MluI, NaeI, NruI, PmaCI, Psp1406I, PvuI, SalI, and SnaBI;
(iii) restriction enzyme BstUI, and (iv) adding restriction enzyme reaction buffer and sealing;
(b) treating the reaction vessel with a restriction enzyme at a temperature of 30 to 50° C. for 0.5 to 16 hours, and then at 50 to 70° C. for 0.5 to 20 hours;
(c) amplifying a part or all of the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start site of the gene to be measured using the DNA treated with the restriction enzyme in step (b) as a template;
(d) measuring the methylation level of CpG in the DNA of the transcriptional regulatory region and/or the region of the transcription start site between +1 and +1000, which were amplified in step (c), based on the results of the amplification in step (c);
The method for measuring the methylation level of CpG in the transcriptional regulatory region and/or the region of the transcriptional start site of a gene to be measured, comprising steps (a) to (d), characterized in that the DNA in the transcriptional regulatory region and/or the region of the transcriptional start site to be amplified in step (c) contains one or more sequences recognized by the restriction enzyme used in step (a).
[2] The method for measuring the methylation level of CpG according to [1] above, characterized in that the DNA in the transcriptional regulatory region and/or the region from +1 to +1000 of the transcription start site to be amplified in step (c) contains a sequence recognized by the restriction enzyme used in step (a) at 1 to 4 positions in total.
[3] The method for measuring a methylation level of CpG according to [1] or [2] above, wherein, in the case where two or more kinds of restriction enzymes are used in step (a), the DNA in the transcriptional regulatory region and/or the region from +1 to +1000 of the transcription start site to be amplified in step (c) does not contain a sequence recognized by at least one of the restriction enzymes used in step (a).
[4] The method for measuring a methylation level of CpG according to [1] or [2] above, characterized in that in step (a), at least HhaI, HpaII and BstUI are added to the reaction vessel as restriction enzymes.
[5] The method for measuring a CpG methylation level according to [1] or [2] above, characterized in that the gene to be measured is the SEPT9 gene.
[6] The method for measuring a methylation level of a CpG according to [5] above, wherein the cytosine of the CpG whose methylation level is measured in step (d) is the cytosine at position 75,369,591, 75,369,593, 75,369,600 or 75,369,602 of human chromosome 17.
[7] The method for measuring a CpG methylation level according to [1] or [2] above, wherein the gene to be measured is the Homeobox A1 (HOXA1) gene.
[8] The method for measuring the methylation level of CpG according to [7] above, characterized in that the cytosine of the CpG whose methylation level is measured in step (d) is the cytosine at position 27,095,945 of human chromosome 7.
[9] The method for measuring the CpG methylation level according to [1] or [2] above, characterized in that in step (c), a part or all of the region of exon 1 of the gene to be measured is amplified.
[10] The method for measuring a methylation level of CpG according to [1] or [2] above, characterized in that in step (a), exonuclease I (ExoI) is further added to the reaction vessel.
[11] The method for measuring a CpG methylation level according to [1] or [2] above, characterized in that the DNA containing the gene to be measured is DNA extracted from plasma or serum.
[12] The method for measuring the methylation level of CpG according to [1] or [2] above, characterized in that no sodium bisulfite treatment is performed.

The present invention makes it possible to easily and quickly measure the CpG methylation level of the gene being measured.

1 is a graph showing the relationship between the number of methylated SEPT9 genes (copy numbers) obtained by a single treatment (condition 1) and the copy numbers obtained by a two-step treatment (condition 2). 1 is a graph showing the relationship between the number of methylated HOXA1 genes (copy numbers) obtained by a single treatment (condition 1) and the copy numbers obtained by a two-step treatment (condition 2).

The method for measuring the CpG methylation level of a gene to be measured according to the present invention comprises the steps of:
(a) placing in a reaction vessel: (i) DNA encoding a gene to be measured;
(ii) at least one restriction enzyme selected from HhaI, HpaII, Hpy99I, SacII, SmaI, NotI, AatII, AccI, Aor13HI, Aor51HI, BspT104I, BssHII, Cfr10I, ClaI, CpoI, Eco52I, HaeII, MluI, NaeI, NruI, PmaCI, Psp1406I, PvuI, SalI, and SnaBI;
(iii) restriction enzyme BstUI, and (iv) adding restriction enzyme reaction buffer and sealing;
(b) treating the reaction vessel with a restriction enzyme at a temperature of 30 to 50° C. for 0.5 to 16 hours, and then at 50 to 70° C. for 0.5 to 20 hours;
(c) amplifying a part or all of the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start site of the gene to be measured using the DNA treated with the restriction enzyme in step (b) as a template;
(d) measuring the methylation level of CpG in the DNA of the transcriptional regulatory region and/or the region from +1 to +1000 of the transcription start site amplified in step (c);
The present invention is not particularly limited as long as it is a method for measuring the methylation level of CpG in the transcriptional regulatory region and/or the region +1 to +1000 of the transcription start site of a gene to be measured, comprising steps (a) to (d), characterized in that the DNA in the transcriptional regulatory region and/or the region +1 to +1000 of the transcription start site amplified in step (c) contains one or more sequences recognized by the restriction enzyme used in step (a), and hereinafter, is also referred to simply as the "method for measuring the methylation level of the present CpG."

In this specification, CpG methylation refers to a state in which a methyl group is added to the carbon atom at the 5th position of the cytosine (C) residue in a CpG sequence, which is a two-base sequence (dinucleotide) in which a guanine (G) base appears next to a cytosine (C) base in DNA. Here, the "p" above represents the phosphodiester bond between cytosine and guanine.

The CpG methylation level in this specification can be evaluated by the number of DNA fragments in which the cytosines in the CpGs are methylated (copy number) when a specific region of the DNA encoding the gene to be measured is amplified to obtain DNA fragments. Specifically, examples of the copy number include the copy number per unit amount of the solution treated with the restriction enzyme in step (c), for example, per 1 μL, the copy number per total amount of the solution treated with the restriction enzyme in step (c), and the copy number per unit amount of the DNA source specimen (for example, per mL of serum or per μg of stool DNA).

In this specification, the transcriptional regulatory region of a gene refers to a region that contains a nucleic acid sequence to which a transcriptional regulatory factor binds and that regulates the amount of transcription of the gene. This transcriptional regulatory region includes promoter regions, enhancer regions, suppressor regions, etc.

In this specification, the transcription start point refers to the site where transcription of mRNA begins, and corresponds to the position corresponding to the first base of mRNA. In this specification, the transcription start point is represented as "+1," and the position one base upstream of the transcription start point is represented as "-1." Therefore, +1000 of the transcription start point means the position 999 bases downstream when the transcription start point is +1.

In addition, the region of +1 to +1000 from the transcription start point in this specification includes exons and introns within the region of +1 to +1000 from the transcription start point, and examples of exons include exon 1, exon 2, and exon 3, and examples of introns include intron 1 and intron 2. Furthermore, examples of the region of +1 to +1000 from the transcription start point include the region of +1 to +500 from the transcription start point, the region of +1 to +350 from the transcription start point, the region of +1 to +100 from the transcription start point, and the region of +1 to +60 from the transcription start point.

In addition, the region +1 to +1000 from the transcription start point in this specification can refer to a region that is +1 to +1000 from the transcription start point and is 100 bases upstream to 100 bases downstream from the adenine (A) position of the start codon, preferably a region that is 50 bases upstream to 50 bases downstream from the start codon. Here, the start codon refers to the codon that serves as the start point of protein synthesis when mRNA is translated into protein.

<Step (a)>
The reaction vessel in this specification is not particularly limited as long as it is a vessel capable of restriction enzyme treatment and is sealable. Here, "sealed" means a state in which the liquid in the reaction vessel cannot leak out, and "sealed" means, for example, putting a lid on a vessel for holding liquid, such as a microtube.

The gene to be measured in this specification is not particularly limited, so long as it contains one or more sequences recognized by the restriction enzyme used in step (a) in the transcriptional regulatory region and/or the region from +1 to +1000 of the transcription start point of the gene. Specifically, the recognition sequences of the four bases are HhaI (GCG/C), HpaII (C/CGG), and BstUI (CG/CG), the recognition sequence of the five bases is Hpy99I (CGWCG/), the recognition sequence of the six bases is SacII (CCGC/GG), SmaI (CCC/GGG), AatII (GACGT/C), AccI (GT/MKAC), Aor13HI (T/CCGGA), Aor51HI (AGC/GCT), BspT104I (TT/CGAA), BssHII (G/CGCGC), Cfr10I (R/CCGGY), ClaI (AT/CGAT), and Eco52I (C/G Examples of such a gene include genes containing one or more sequences recognized by at least one, and preferably two, methylation-sensitive restriction enzymes selected from the group consisting of CpoI (CG/GWCCG), HaeII (RGCGC/Y), MluI (A/CGCGT), NaeI (GCC/GGC), NruI (G/CTAGC), PmaCI (CAC/GTG), Psp1406I (AA/CGTT), PvuI (CGAT/CG), SalI (G/TCGAC), SnaBI (TAC/GTA), CpoI (CG/GWCCG), which has a seven-base recognition sequence, and NotI (GC/GGCCGC), which has an eight-base recognition sequence. Furthermore, the transcriptional regulatory region and/or the region from +1 to +1000 of the transcriptional start site of the gene may contain one or more, preferably one or two, sequences recognized by the restriction enzyme described in step (a)(ii), and one or more, preferably one, sequences recognized by BstUI. Note that the above "W" means A or T, "M" means A or C, "K" means G or T, "R" means A or G, and "Y" means C or T.

Specific examples of genes to be measured include the SEPT9 gene, HOXA1 gene, SST gene, RUNX3 gene, ADAMTS2 gene, PCDH10 gene, SEMA5A gene, SPSB4 gene, BMP3 gene, NDRG4 gene, and SDC2 gene.

The DNA containing the gene to be measured can be obtained by extraction from biological samples such as plasma, serum, blood cells, stool, blood, saliva, urine, hair roots, tears, nasal discharge, pleural fluid, ascites, cerebrospinal fluid, sputum, vaginal secretions, and tissues, and is preferably DNA extracted from plasma or serum. Methods for extracting DNA from biological samples include phenol extraction, phenol-chloroform extraction, alkaline dissolution, and the like, as well as methods using commercially available DNA extraction reagents.

The restriction enzymes to be added to the reaction vessel include (ii) at least one, preferably two or more, more preferably two restriction enzymes selected from HhaI, HpaII, Hpy99I, SacII, SmaI, NotI, AatII, AccI, Aor13HI, Aor51HI, BspT104I, BssHII, Cfr10I, ClaI, CpoI, Eco52I, HaeII, MluI, NaeI, NruI, PmaCI, Psp1406I, PvuI, SalI, and SnaBI, and (iii) BstUI. Note that BstUI is also sold under the names AccII, Bsh1236I, BspFNI, BstFNI, FnuDII, or MvnI, depending on the manufacturer of the restriction enzyme.

The above restriction enzyme is a methylation-sensitive restriction enzyme. A methylation-sensitive restriction enzyme is a restriction enzyme that can distinguish between methylated and unmethylated CpG in a sequence that the restriction enzyme recognizes. Note that methylation-sensitive restriction enzymes are used in a method for analyzing CpG methylation, and utilize the phenomenon that cytosine in a sequence that the restriction enzyme recognizes becomes methylated and the enzyme is unable to cleave DNA. Preferred combinations of the above restriction enzymes include two types, HhaI and BstUI, HpaII and BstUI, and three types, HhaI, HpaII and BstUI. Note that it is preferable to use heat-inactivated BstUI, specifically BstUI that remains active even after treatment at 65°C for 20 minutes (or treatment at 60°C for 1 hour).

When treating with the above-mentioned methylation-sensitive restriction enzymes, if single-stranded DNA is present in the template DNA, the single-stranded DNA will avoid cleavage by the restriction enzyme and will be amplified in the subsequent PCR reaction, causing a false positive. To avoid this, it is preferable to add exonuclease I (ExoI) to remove single-stranded DNA.

The restriction enzyme reaction buffer may be any buffer capable of cleaving DNA by the enzymatic reaction of the restriction enzyme used, and may be selected appropriately depending on the restriction enzyme used. In addition to commercially available restriction enzyme reaction buffers, PCR buffers may also be used.

<Step (b)>
Step (b) is a step of performing restriction enzyme treatment. The treatment with the restriction enzyme has two temperature stages, and the treatment temperature in the first stage can be appropriately adjusted, but is, for example, within the range of 30 to 50° C., preferably within the range of 35 to 40° C., and more preferably within the range of 36 to 38° C. The treatment time in the first stage can be appropriately adjusted, but is, for example, within the range of 0.5 to 16 hours, preferably 0.7 to 8 hours, more preferably 0.9 to 4 hours, and even more preferably 1 to 2 hours.

The processing temperature in the second stage can be adjusted as appropriate, but is, for example, within the range of 50 to 70°C, preferably within the range of 55 to 65°C, and more preferably within the range of 58 to 62°C. The processing time in the second stage can be adjusted as appropriate, but is, for example, within the range of 0.5 to 20 hours, preferably within the range of 6 to 18 hours, more preferably within the range of 12 to 17 hours, and even more preferably within the range of 14 to 16 hours.

The restriction enzyme treatment is carried out in succession in the first and second stages while the reaction vessel is sealed. In other words, the second stage is carried out without releasing the sealed state, such as by opening the lid of the reaction vessel after the first stage. In conventional methods, such as the method described in Patent Document 2, the lid of the reaction vessel is opened after the first stage, a restriction enzyme is added, the lid is closed and sealed, and the second stage is carried out. Therefore, the work of adding the restriction enzyme in the second stage is complicated and requires time and effort. There is also the problem that the liquid in the reaction vessel evaporates, changing the composition in the reaction vessel. On the other hand, in the present method for measuring the methylation level of CpG, all of the restriction enzymes are added to the reaction vessel at once, so there is no need to open the lid of the reaction vessel after the first stage.

<Step (c)>
In step (c), the DNA treated with the restriction enzyme in step (b) is used as a template to amplify a part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start site of the gene to be measured. The part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start site of the gene to be measured to be amplified may be a region containing at least one sequence recognized by the restriction enzyme used in step (a), and may be a region containing one to four sequences, preferably one to three sequences. Furthermore, in the part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start site of the gene to be measured to be amplified, there may be one, two, three, or four sequences recognized by one type of methylation-sensitive restriction enzyme used in step (a). Furthermore, when two or more types of restriction enzymes are used in step (a), any one of the restriction enzymes may recognize only one sequence, or one type of restriction enzyme may recognize one sequence and another type of restriction enzyme may recognize two or more sequences, preferably two sequences. In addition, the region to be amplified may be a part of the transcriptional regulatory region of the gene to be measured and a part of the region from +1 to +1000 of the transcription start site, only a part of the transcriptional regulatory region of the gene to be measured, only a part of the region from +1 to +1000 of the transcription start site of the gene to be measured, or only a part of the transcriptional regulatory region of the gene to be measured and a part of the region of exon 1.

The length of the transcriptional regulatory region of the gene to be measured and/or the part or whole of the region between +1 and +1000 of the transcription start site to be amplified can be 30 to 1000 bases, preferably 40 to 200 bases, and more preferably 50 to 150 bases.

When two or more types of restriction enzymes are used in step (a), the region may not contain a sequence recognized by at least one of the restriction enzymes used in step (a) in part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start point of the gene to be amplified. For example, three types of restriction enzymes, X, Y, and Z, may be used in step (a), and the region may contain one or more sequences recognized by the restriction enzymes X and Y independently in part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start point of the gene to be amplified, but may not contain a sequence recognized by the restriction enzyme Z. In the above example, the restriction enzyme Z does not affect the measurement of methylation and is therefore unnecessary. However, by cutting the DNA even a little in the restriction enzyme treatment in step (b), the PCR amplification efficiency can be improved, which is useful for measuring methylation.

The method for amplifying part or all of the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start site of the gene to be measured is not particularly limited, but examples include PCR methods such as digital PCR (polymerase chain reaction) and real-time PCR; LAMP (Loop-Mediated Isothermal Amplification); etc.

The "digital PCR" is a method for absolute quantitative determination of the amount of sample DNA. In this method, the sample DNA is partitioned (diluted and distributed) into approximately 20,000 droplets or wells, and PCR is performed using a thermal cycler. The droplets and wells that contain the target DNA glow, while those that do not glow do not change. The principle is that the concentration of the gene to be measured in the sample can be obtained as an absolute value by counting the number of glowing droplets or wells and non-glowing droplets or wells. The data is determined by the presence/absence of the target DNA in the droplets or wells, which is the same as a digital signal of 1/0, so it is called "digital PCR". In addition, the "copy number count" in digital PCR refers to the process of measuring the amount of fluorescence of the approximately 20,000 droplets and wells mentioned above one by one using a droplet reader, counting the number of droplets and wells that emit fluorescence and the number of droplets and wells with weak fluorescence intensity, and measuring the absolute value of the copy number of the gene by correction using a Poisson model.

Specifically, the digital PCR is carried out as follows.
(1) A PCR reaction solution containing template DNA is compartmentalized into approximately 20,000 droplets.
(2) Approximately 20,000 droplets are collected into one tube.
(3) Perform PCR reaction (PCR will be performed on approximately 20,000 droplets per tube).
(4) The fluorescence intensity of approximately 20,000 droplets is measured one by one, the number of droplets with strong fluorescence intensity and those with weak fluorescence intensity are counted, and the copy number of the target gene in the sample is calculated by correction using a Poisson model. Here, the strong fluorescence intensity of a certain droplet means that the target gene is contained in that droplet and the fluorescence intensity has been increased by PCR amplification.

Examples of the PCR method include the TaqMan probe method, the intercalator method, the 5'-nuclease method, and the cycling probe method.

When the gene to be measured is the SEPT9 gene, a part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start point of the SEPT9 gene can specifically be the sequence of the region from the transcription start point (position 75,369,272 of human chromosome 17) to +295 to +356 of SEPT9 gene transcript variant 2 (positions 75,369,566 to 75,369,627 of human chromosome 17 in the position information in the GRCh37/hg19 database: SEQ ID NO: 1). The sequence of the +295 to +356 region corresponds to the exon 1 region. Furthermore, when the gene to be measured is the SEPT9 gene, examples of the cytosine of the CpG whose methylation level is measured in step (d) include the cytosine at positions 75,369,591, 75,369,593, 75,369,600, or 75,369,602 of human chromosome 17 (the cytosines at positions 26, 28, 35, and 37 in SEQ ID NO: 1);
positions 75,369,591 and 75,369,593 of human chromosome 17;
positions 75,369,591 and 75,369,600 on human chromosome 17;
positions 75,369,591 and 75,369,602 of human chromosome 17;
positions 75,369,593 and 75,369,600 on human chromosome 17;
two cytosines at positions 75,369,593 and 75,369,602 of human chromosome 17, or at positions 75,369,600 and 75,369,602 of human chromosome 17;
positions 75,369,591, 75,369,593, and 75,369,600 of human chromosome 17;
positions 75,369,591, 75,369,593, and 75,369,602 of human chromosome 17;
three cytosines at positions 75,369,591, 75,369,600, and 75,369,602 of human chromosome 17, or at positions 75,369,593, 75,369,600, and 75,369,602 of human chromosome 17;
and four cytosines at positions 75,369,591, 75,369,593, 75,369,600, and 75,369,602 of human chromosome 17.

When the gene to be measured is the HOXA1 gene, the transcriptional regulatory region and/or part or all of the region from +1 to +1000 of the transcription start point of the HOXA1 gene can specifically be the sequence of the region from the transcription start point of the HOXA1 gene (position 27,096,000 of human chromosome 7) to +20 to +127 (positions 27,095,874 to 27,095,981 of human chromosome 7 in the location information in the GRCh38/hg38 database: SEQ ID NO: 2). The sequence of the +20 to +127 region corresponds to the exon 1 region. Furthermore, when the gene to be measured is the HOXA1 gene, the cytosine of the CpG whose methylation level is measured in step (d) can be the cytosine at position 27,095,945 of human chromosome 7 in the exon 1 region (the 37th cytosine in SEQ ID NO: 2).

As the primers for amplifying a part or all of the region +1 to +1000 of the transcription regulatory region and/or transcription start point of the gene to be measured, and the probe for detecting the amplified nucleic acid, a primer set, probe, or a label thereof for individually measuring the methylation of each cytosine of the CpG to be measured contained in a part or all of the region +1 to +1000 of the transcription regulatory region and/or transcription start point of the gene to be measured may be used, but it is preferable to use a primer set, probe, or a label thereof for commonly measuring the methylation of each cytosine of the CpG to be measured in the DNA of a part or all of the region +1 to +1000 of the transcription regulatory region and/or transcription start point to be amplified. Specifically, for example, when two CpGs to be measured are contained in a part or all of the region +1 to +1000 of the transcription regulatory region and/or transcription start point of the gene to be measured, the primers may be two pairs of primers for amplifying each CpG individually, or one pair of primers that can amplify two CpGs at once. In addition, the probes may be two probes that hybridize to the base sequences of the respective PCR amplified regions, or one probe that hybridizes to the base sequences of both PCR amplified regions. Note that the CpG in steps (c) and (d) above is a CpG in a sequence that is recognized by at least one of the restriction enzymes used in step (a).

When the gene to be measured is SEPT9, suitable examples include a forward primer consisting of the base sequence shown in SEQ ID NO:3, or a forward primer consisting of the base sequence shown in SEQ ID NO:3 with one or several bases substituted, deleted, inserted or added; a reverse primer consisting of the base sequence shown in SEQ ID NO:4, or a reverse primer consisting of the base sequence shown in SEQ ID NO:4 with one or several bases substituted, deleted, inserted or added; a probe consisting of the base sequence shown in SEQ ID NO:5, or a probe consisting of the base sequence shown in SEQ ID NO:5 with one or several bases substituted, deleted, inserted or added; and these labels.

When the gene to be measured is HOXA1, suitable examples include a forward primer consisting of the base sequence shown in SEQ ID NO:6, or a forward primer consisting of the base sequence shown in SEQ ID NO:6 in which one or several bases have been substituted, deleted, inserted or added; a reverse primer consisting of the base sequence shown in SEQ ID NO:7, or a reverse primer consisting of the base sequence shown in SEQ ID NO:7 in which one or several bases have been substituted, deleted, inserted or added; a probe consisting of the base sequence shown in SEQ ID NO:8, or a probe consisting of the base sequence shown in SEQ ID NO:8 in which one or several bases have been substituted, deleted, inserted or added; and these labels.

The above "base sequence in which one or several bases have been substituted, deleted, inserted, or added" refers to a base sequence in which, for example, 1 to 5 bases, preferably 1 to 3 bases, and more preferably 1 to 2 bases have been substituted, deleted, inserted, or added.

Labeling substances for the forward primer label, reverse primer label, and probe label include enzymes such as peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride, and tetramethylrhodamine isothiocyanate; green fluorescent protein (GFP), cyan fluorescent protein (Cyan Fluorescence Protein), and the like. Examples of reporter fluorescent substances include fluorescent proteins such as 5-fluorouracil (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and luciferase; radioisotopes such as 3H, 14C, 125I, and 131I; biotin, avidin, or chemiluminescent substances; and combinations of reporter fluorescent substances and quencher substances or structures (for example, a combination of 5-FAM [5-Carboxyfluorescein] and 5-TAMRA [5-Carboxytetramethylrhodamine], a combination of VIC and MGB [Minor Groove Binder], and a combination of 5-FAM and MGB).

<Step (d)>
In step (d), the methylation level of CpG in DNA in the region of +1 to +1000 of the transcription regulatory region and/or transcription start site targeted for amplification in step (c) is measured based on the results of the amplification in step (c). Specifically, the methylation level of the CpG can be measured based on the degree of amplification of DNA that has escaped cleavage by a methylation-sensitive restriction enzyme due to methylation in step (c). The degree of DNA amplification can be evaluated by detecting the amplified DNA with a probe or the like.

In addition, in step (d), "measuring the methylation level of CpGs" may involve not only measuring the methylation of CpGs in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site to be amplified, but also measuring the methylation of the corresponding CpGs in the complementary strand.

In the present method for measuring the methylation level of CpG, it is preferable not to perform sodium bisulfite treatment. Bisulfite treatment processes single-stranded DNA, which easily decomposes the DNA, and approximately 90% of the DNA is denatured and lost. Therefore, since the detection limit of quantitative PCR is approximately 10 copies, more than 100 copies of target DNA are required for sodium bisulfite treatment. When quantitatively analyzing the absolute copy number using quantitative PCR, it is necessary to create a calibration curve using a standard substance, but there is a problem that the reliability of the measured values for low copy numbers is extremely low. On the other hand, digital PCR does not require the creation of a calibration curve, and accurate absolute number measurement is possible even at low copy numbers.

The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

(Preparation of SEPT9 or HOXA1 methylation control DNA samples)
As a control for the methylated alleles of SEPT9 and HOXA1, EpiScope Methylated HCT116 gDNA (DNA derived from the human colon cancer cell line HCT116 that is highly methylated by CpG methylase, and is a SEPT9 or HOXA1 methylation control DNA) was used, which is known to have high levels of methylation of various genes including SEPT9 and HOXA1.

(Preparation of unmethylated control DNA samples of SEPT9 or HOXA1)
As a control for the SEPT9 or HOXA1 unmethylated allele, DNA derived from peripheral blood lymphocytes (SEPT9 unmethylated control DNA or HOXA1 unmethylated control DNA) known to have almost no methylation of SEPT9 or HOXA1 was used. Using a blood collection tube containing the anticoagulant EDTA-2Na, 0.2% NaCl was added to the collected peripheral blood, which was then mixed by inversion and centrifuged at 2500 rpm for 5 minutes. The supernatant was removed with an aspirator so as not to suck up the white blood cells precipitated at the bottom of the tube, and this operation was repeated until the pellet (white blood cells) turned white. The whitened pellet was transferred to a 1.5 mL tube, and nucleic acid extraction was performed using the nucleic acid extraction agent SepaGene (Sanko Junyaku Co., Ltd.). The extraction method followed the instructions for use. The nucleic acid pellet obtained by extraction was air-dried and dissolved in 100 μL of TE buffer to prepare a SEPT9 unmethylated control DNA or HOXA1 unmethylated control DNA sample.

Next, methylated control DNA (EpiScope Methylated HCT116 gDNA) was added to a fixed concentration (0.8 ng/μL) of unmethylated control DNA to prepare a dilution series of methylated control DNA samples (0%, 3.1%, 6.3%, 12.5%, 25%, 50%, 100%).

Table 1 shows the relationship between the concentration and dilution series (%) of the methylation control DNA (SEPT9 methylation control or HOXA1 methylation control) added to each sample.

(Enzyme treatment)
Next, an enzyme treatment containing a methylation-sensitive restriction enzyme was carried out under the following two conditions. Condition 2 is the method described in Patent Document 1 above.

<Condition 1: Single treatment> One-step enzyme treatment of DNA: simultaneous treatment with HhaI, HpaII, ExoI, and BstUI 1. Place 10 μL of the DNA sample prepared according to Table 1 in a 1.5 mL tube.
2. Add 1 μL of AmpliTaq Gold buffer II (AmpliTaq Gold DNA polymerase kit N808-0241: Thermo Fisher Scientific).
Add 1 μL of 3.25 mM _MgCl2 solution (included in AmpliTaq Gold DNA Polymerase Kit N808-0241: Thermo Fisher Scientific).
4. Add 1 μL (10 U) of HhaI (Thermo Fisher Scientific).
5. Add 1 μL (10 U) of HpaII (Thermo Fisher Scientific).
6. Add 1 μL (20 U) of ExoI (Thermo Fisher Scientific).
7. Add 1 μL (10 U) of BstUI (New England BioLabs).
8. Mix by pipetting and then close the tube with the lid to seal.
9. Heat to 37 degrees for 1 hour.
10. Heat to 60 degrees for 16 hours.
11. Heat to 98 degrees for 10 minutes.

<Condition 2: Two-step treatment> Two-step enzyme treatment of DNA: treatment with HhaI, HpaII, and ExoI, followed by treatment with BstUI. 1.1. Place 10 μL of the DNA sample prepared according to Table 1 in a 5 mL tube.
2. Add 1 μL of AmpliTaq Gold buffer II (AmpliTaq Gold DNA polymerase kit N808-0241: Thermo Fisher Scientific).
Add 1 μL of 3.25 mM _MgCl2 solution (included in AmpliTaq Gold DNA Polymerase Kit N808-0241: Thermo Fisher Scientific).
4. Add 1 μL (10 U) of HhaI (Thermo Fisher Scientific).
5. Add 1 μL (10 U) of HpaII (Thermo Fisher Scientific).
6. Add 1 μL (20 U) of ExoI (Thermo Fisher Scientific).
7. Mix by pipetting and then close the tube with the lid to seal.
8. Heat to 37 degrees for 16 hours.
9. Open the cap of the tube and add 1 μL (10 U) of BstUI (New England BioLabs), then close the cap of the tube and seal it.
10. Heat to 60 degrees for 16 hours.
11. Heat to 98 degrees for 10 minutes.

The above HhaI, HpaII, and BstUI are methylation-sensitive restriction enzymes that recognize 5'-GCGC-3', 5'-CCGG-3', and 5'-CGCG-3', respectively, and cleave the base sequence. If the cytosine of the CpG in this base sequence is methylated, this base sequence will remain uncleaved, making it possible to amplify it in the subsequent PCR.

Table 2 shows the base sequence of the region to be amplified by PCR. In Table 2, the underlined bases in the base sequence column of the region to be amplified by PCR indicate sites that may be cut by the restriction enzymes HhaI, HpaII, or BstUI. However, since these restriction enzymes are methylation-sensitive restriction enzymes, if the cytosine (C) of the underlined CG in Table 2 is methylated, the recognition site of the above restriction enzymes containing the methylated cytosine will not be cut. Furthermore, each amplified region is part of the exon 1 region. In addition, the RefSeq of SEPT9 mRNA in the NCBI Reference Sequence Database is NM_001113493 (updated June 14, 2013), and the RefSeq of HOXA1 mRNA is NM_005522 (updated February 17, 2023) or NM_153620 (updated February 17, 2023), and the transcription start point of HOXA1 in this specification is represented based on NM_005522.

(Digital PCR processing)
As described above, "digital PCR" is a method for absolutely quantifying the amount of DNA in a sample. Here, a digital PCR system (QX200 Droplet Digital PCR System: Bio-Rad) was used.

After the enzyme reaction was completed, 2 μL of the enzyme-reacted DNA solution treated under Condition 1 or Condition 2 above, 6 μL of water, and 14 μL of PCR reaction solution were mixed. This PCR reaction solution consisted of 1 μL each of 20 μM primer mix and 5 μM probe (total 4 μL) for amplifying a specific region of the SEPT9 gene or HOXA1 gene and measuring a specific methylated cytosine, and 10 μL of 2x ddPCR master mix (Bio-Rad). Multiplex PCR was performed using the above DNA-containing PCR reaction solution.

The base sequences of the primer and probe for SEPT9 are as follows:
Forward primer:
5'-GCCCACCAGCCATCATGT-3' (SEQ ID NO: 3)
Reverse primer:
5'-GTCCGAAATGATCCCCATCCA-3' (SEQ ID NO: 4)
Probe:
5'-FAM-CCGCGGTCAACGC-MGB-3' (SEQ ID NO:5)

The base sequences of the primers and probe for HOXA1 are as follows:
Forward primer:
5'-CCCATGGAGGAAGTGAGAAA-3' (SEQ ID NO: 6)
Reverse primer:
5'-GGGGTATTCCAGGAAGGAGT-3' (SEQ ID NO: 7)
Probe:
5'-FAM-GCACAGTCACGCCGG-MGB-3' (SEQ ID NO: 8)

Droplets were made from this DNA-containing PCR reaction solution using an Automated Droplet Generator (Bio-Rad). Using a thermal cycler, the droplets were preheated at 95°C for 10 minutes, and then the DNA was amplified by repeating 40 cycles of thermal denaturation at 94°C for 30 seconds and annealing at 56°C for 60 seconds, and finally heated at 98°C for 10 minutes. After DNA amplification, the fluorescent dye derived from the TaqMan probe in each droplet was detected using a QX200 Droplet Reader (Bio-Rad) and QuantaSoft software (Bio-Rad), and the number of DNA fragments (copy numbers) in which cytosines in CpGs in the amplified region of the gene to be measured were methylated, and which had escaped cleavage by methylation-sensitive restriction enzymes, was measured.

The base sequence (5'-GCGC-3', 5'-CCGG-3', 5'-CGCG-3') containing the underlined CpG in Table 2 of DNA in the amplification target region of SEPT9 or HOXA1 can be recognized by any of the three restriction enzymes used (HhaI, HpaII, BstUI). For example, in SEPT9, if the CpGs of 5'-GCGC-3' and/or 5'-CGCG-3' are not methylated, they are recognized and cleaved by the restriction enzymes HhaI and/or BstUI, and are not amplified by PCR, so they are not detected. On the other hand, if all the CpGs of 5'-GCGC-3' and 5'-CGCG-3' are methylated, they escape cleavage by the restriction enzymes and are amplified by PCR, so they are detected. Therefore, when all the cytosines of the CpGs in the base sequences GCGC and CGCG in the region to be amplified by PCR of SEPT9 in the template DNA shown in Table 2 are methylated, cleavage of GCGC and CGCG does not occur even when restriction enzyme treatment is performed in a single process (condition 1) or in a two-step process (condition 2), and the region can be amplified by subsequent PCR, so that the number of copies of methylated SEPT9 can be counted by digital PCR.
In HOXA1, if the CpG at 5'-CCGG-3' is not methylated, it is recognized by HpaII, cleaved, and not amplified by PCR, and therefore not detected. On the other hand, if the CpG at 5'-CCGG-3' is methylated, it escapes cleavage by the restriction enzyme and is amplified by PCR, and therefore is detected. Therefore, if the cytosine of the CpG in the base sequence CCGG in the PCR amplification target region of HOXA1 in the template DNA shown in Table 2 is methylated, cleavage of CCGG does not occur even if restriction enzyme treatment is performed by single treatment (condition 1) or two-step treatment (condition 2), and the region can be amplified by subsequent PCR, so that it can be counted as the number of methylated HOXA1 copies by digital PCR.
The amplified region of SEPT9 contains the BstUI and HhaI cleavage sites but not the HpaII cleavage site, and the amplified region of HOXA1 contains the HpaII cleavage site but not the BstUI and HhaI cleavage sites.

(result)
Comparison of copy numbers of methylated SEPT9 or methylated HOXA1 due to differences in restriction enzyme treatment In the SEPT9 gene, a graph showing the relationship between the methylated SEPT9 copy number obtained by a single treatment (condition 1) and the methylated SEPT9 copy number obtained by a two-step treatment (condition 2) is shown in FIG. 1, and similarly, in the HOXA1 gene, a graph showing the relationship between the methylated HOXA1 copy number obtained by a single treatment (condition 1) and the methylated HOXA1 copy number obtained by a two-step treatment (condition 2) is shown in FIG. 2. The copy number of SEPT9 when 0%, 3.1%, 6.3%, 12.5%, 25%, 50%, and 100% methylation control DNA dilution series samples were treated with restriction enzymes under condition 2 is shown on the horizontal axis, and the copy number of SEPT9 when treated with restriction enzymes under condition 1 is shown on the vertical axis. As a result of analysis by linear regression analysis, Y = 1.08X in FIG. 1 and Y = 0.99X in FIG. 2.

These results confirmed a correlation between restriction enzyme treatment under condition 2 and restriction enzyme treatment under condition 1. Therefore, it was confirmed that the present method for measuring the methylation level of CpG can easily measure the same level of methylation as that obtained by conventional restriction enzyme treatment.

Claims (12)

(a) placing in a reaction vessel: (i) DNA encoding a gene to be measured;
(ii) at least one restriction enzyme selected from HhaI, HpaII, Hpy99I, SacII, SmaI, NotI, AatII, AccI, Aor13HI, Aor51HI, BspT104I, BssHII, Cfr10I, ClaI, CpoI, Eco52I, HaeII, MluI, NaeI, NruI, PmaCI, Psp1406I, PvuI, SalI, and SnaBI;
(iii) restriction enzyme BstUI, and (iv) adding restriction enzyme reaction buffer and sealing;
(b) treating the reaction vessel with a restriction enzyme at a temperature of 30 to 50° C. for 0.5 to 16 hours, and then at 50 to 70° C. for 0.5 to 20 hours;
(c) amplifying a part or all of the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start site of the gene to be measured using the DNA treated with the restriction enzyme in step (b) as a template;
(d) measuring the methylation level of CpG in the DNA of the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start site that was the target of amplification based on the results of the amplification in step (c);
The method for measuring the methylation level of CpG in the region of the transcriptional regulatory region and/or the transcriptional start site of a gene to be measured, comprising steps (a) to (d), characterized in that the DNA in the region of the transcriptional regulatory region and/or the transcriptional start site of the gene to be amplified in step (c) contains one or more sequences recognized by the restriction enzyme used in step (a). The method for measuring the methylation level of CpG according to claim 1, characterized in that the DNA in the transcriptional regulatory region and/or the region +1 to +1000 of the transcription start site targeted for amplification in step (c) contains a sequence recognized by the restriction enzyme used in step (a) at a total of 1 to 4 locations. The method for measuring the methylation level of CpG according to claim 1 or 2, characterized in that when two or more types of restriction enzymes are used in step (a), the DNA in the transcriptional regulatory region and/or the region +1 to +1000 of the transcription start site to be amplified in step (c) does not contain a sequence recognized by at least one of the restriction enzymes used in step (a). The method for measuring the methylation level of CpG according to claim 1 or 2, characterized in that in step (a), at least HhaI, HpaII and BstUI are added as restriction enzymes to the reaction vessel. The method for measuring the methylation level of CpG according to claim 1 or 2, characterized in that the gene to be measured is the SEPT9 gene. The method for measuring the methylation level of CpG according to claim 5, characterized in that the cytosine of the CpG whose methylation level is measured in step (d) is the cytosine at position 75,369,591, 75,369,593, 75,369,600 or 75,369,602 of human chromosome 17. The method for measuring the methylation level of CpG according to claim 1 or 2, characterized in that the gene to be measured is the Homeobox A1 (HOXA1) gene. The method for measuring the methylation level of a CpG according to claim 7, characterized in that the cytosine of the CpG whose methylation level is measured in step (d) is the cytosine at position 27,095,945 of human chromosome 7. The method for measuring the methylation level of CpG according to claim 1 or 2, characterized in that in step (c), a part or all of the region of exon 1 of the gene to be measured is amplified. The method for measuring the methylation level of CpG according to claim 1 or 2, characterized in that in step (a), exonuclease I (ExoI) is further added to the reaction vessel. The method for measuring the CpG methylation level according to claim 1 or 2, characterized in that the DNA containing the gene to be measured is DNA extracted from plasma or serum. 3. The method for measuring the methylation level of CpG according to claim 1 or 2, characterized in that sodium bisulfite treatment is not performed.