JP4332467B2 - Method for measuring DNA methylation rate - Google Patents

Description

  The present invention relates to a method for measuring a DNA methylation rate. By measuring the DNA methylation rate, for example, the malignancy of cancer cells can be evaluated, or the differentiation state of various cells can be evaluated. The present invention also relates to a method for evaluating the state of a cell.

Genetic information possessed by living organisms is collected in DNA. In general, it is understood that everything is defined by the base sequence of this DNA, but not only that, but in higher animals, the genomic DNA in the body ages. In fact, there are many diseases caused by the methylation of cytosine bases in DNA that change with aging.
It has been demonstrated in mice that demethylation and rejuvenation again when genomic DNA is transferred into an egg have been demonstrated in cloned animal experiments, in which case the telomere length is also restored. .
When the body's genome ages, genes that should work will not work, or genes that should not work will work. In many cancer cells, methylation of the promoter region that controls gene expression occurs, and many of the gene groups that suppress cancer are inactivated.

Thus, methylation of DNA strands is an important index for various diseases including cancer, and is related to the control of gene expression. For example, it is an index for grasping the degree of cell differentiation. At the same time, various measurement methods have been studied so far.
On the other hand, for example, when DNA methylation is measured at a medical site, it is required to obtain a determination result quickly and accurately. However, from this point of view, the conventional methods are not necessarily preferable methods.

Examples of DNA methylation analysis methods that have been carried out to date, particularly 5-methylcytosine analysis methods, include:
(1) A method in which sample DNA is decomposed into single bases by enzymatic treatment, and this is quantified by applying chromatography or ELISA,
(2) Cytosine base in sample DNA is changed to uracil by chemical treatment such as bisulfite ion and hybridizes specifically to methylated DNA or hybridizes specifically to unmethylated DNA. A method for analyzing methylation of a region to which a primer hybridizes depending on the presence or absence of the appearance of a PCR amplification product using a PCR primer
(3) A method of directly sequencing the DNA sequence using DNA treated with bisulfite ion in the same manner as in the method of (2), or (4) Bisulfite ion-PCR-SSCP (single strand conformational polymorphism) Law (Non-Patent Document 1)
It has been known.

  In the method (1), it is necessary to excise and purify a specific region of the gene to be analyzed with a restriction enzyme, and a large amount of DNA as much as 5 to 50 μg is required as the amount of DNA fragment. When DNA is collected from human blood using this method (1) and methylation analysis is performed on a specific DNA fragment, it depends on the molecular size of the fragment to be analyzed. Several tens of liters of blood is required, and for clinical application, the detection sensitivity of the present method must be dramatically increased, which has not been realized at present.

  In the method (2), as in the method (1), a large amount of DNA sample is not necessary, and it can be amplified by PCR and analyzed for methylation. However, on the premise that the methylation site of the DNA to be analyzed has been analyzed in detail in advance, an oligo DNA primer for PCR is designed based on the result. In addition, in this method, only the methylation of several cytosines present in the region where the primer hybridizes is the object of analysis, and the overall methylation rate of the region between the two PCR primers cannot be analyzed. There are drawbacks.

  In the method (3), the methylation analysis for each sample needs to be analyzed by DNA sequence analysis (sequencing), the operation is complicated, the analysis takes a lot of time and labor, and the implementation cost is high. Yes, it is not suitable for comprehensive methylation analysis of genes.

  In the method (4), PCR amplification is carried out using DNA treated with bisulfite ion as a template, and the resulting DNA is analyzed by SSCP electrophoresis. However, it is difficult to predict the behavior of single-stranded DNA in SSCP electrophoresis, and an approximate difference in methylation rate between two samples can be determined, but it is difficult to accurately quantify. Further, in this method, since DNA in polyacrylamide gel is visualized by silver staining or isotope labeling, the operation is complicated and it takes time until analysis.

M. Maekawa et al., “Biochemical and Biophysical Research Communications” (Netherlands), 1999, 262, p. 671-676

  Therefore, an object of the present invention is to eliminate these disadvantages of the prior art, to make any region a target sequence without analyzing the methylation site in advance, and to easily and accurately detect cytosine of DNA. An object of the present invention is to provide a method for measuring a DNA methylation rate, which can measure the methylation rate. Another object of the present invention is to provide a method for evaluating a cell state, which can visually grasp a methylation state and further a cell state.

The subject is (1) a step of treating single-stranded or double-stranded DNA containing a base sequence to be measured with a cytosine modifying agent capable of converting cytosine into another base (hereinafter referred to as a conversion step). ),
(2) Labeled deoxycytidine 5′-triphosphate (dCTP) or labeled deoxyguanosine 5 ′ using either DNA strand of the obtained double-stranded DNA or the obtained single-stranded DNA as a template A step of performing a DNA amplification operation in the presence of triphosphate (dGTP) (hereinafter referred to as an amplification step), and (3) one of the amplified double-stranded DNA strands or the amplified double strand A step of measuring the amount of labeled cytosine or labeled guanine in the double-stranded DNA (hereinafter referred to as analysis step)
It can solve by the method of measuring the methylation rate of cytosine in a measurement object base sequence characterized by including this.

The present invention also includes (1) a step (conversion step) of treating single-stranded or double-stranded DNA containing a measurement target base sequence with a cytosine modifying agent capable of converting cytosine into another base (conversion step),
(2) Labeled deoxy using the obtained double-stranded DNA as a template, the DNA strand (ie, sense strand) containing the converted base sequence derived from the base sequence to be measured, or the obtained single-stranded DNA A step of performing a DNA amplification operation in the presence of cytidine 5′-triphosphate (dCTP) (amplification step); and (3) an amplified base corresponding to the converted base sequence of the amplified double-stranded DNA. A step of measuring the amount of labeled cytosine in a DNA strand containing a sequence (ie, sense strand) or amplified double-stranded DNA (analysis step)
And a method for measuring the methylation rate of cytosine in a base sequence to be measured (hereinafter referred to as the sense strand template-C measurement method of the present invention).

The present invention also includes (1) a step (conversion step) of treating single-stranded or double-stranded DNA containing a measurement target base sequence with a cytosine modifying agent capable of converting cytosine into another base (conversion step),
(2) Labeled deoxy using the obtained double-stranded DNA as a template, the DNA strand (ie, sense strand) containing the converted base sequence derived from the base sequence to be measured, or the obtained single-stranded DNA A step of performing a DNA amplification operation in the presence of guanosine 5′-triphosphate (dGTP) (amplification step), and (3) a post-amplification base corresponding to the post-conversion base sequence of the amplified double-stranded DNA A step of measuring the amount of labeled guanine in the complementary strand (ie, antisense strand) of the DNA strand containing the sequence or in the amplified double-stranded DNA (analysis step)
The method for measuring the methylation rate of cytosine in a measurement target nucleotide sequence (hereinafter referred to as the sense strand template-G type measurement method of the present invention).

The present invention also includes (1) a step (conversion step) of treating single-stranded or double-stranded DNA containing a measurement target base sequence with a cytosine modifying agent capable of converting cytosine into another base (conversion step),
(2) Labeled deoxycytidine 5′-three using as a template the complementary strand (ie, antisense strand) of the obtained double-stranded DNA containing the converted base sequence derived from the base sequence to be measured. A step of performing a DNA amplification operation in the presence of phosphoric acid (dCTP) (amplification step); and (3) a DNA strand comprising an amplified base sequence corresponding to the post-conversion base sequence of the amplified double-stranded DNA. A step of measuring the amount of labeled cytosine in the complementary strand (ie, antisense strand) or amplified double-stranded DNA (analysis step)
The method of measuring the methylation rate of cytosine in a measurement target base sequence (hereinafter referred to as the antisense strand template-C type measurement method of the present invention).

The present invention also includes (1) a step (conversion step) of treating single-stranded or double-stranded DNA containing a measurement target base sequence with a cytosine modifying agent capable of converting cytosine into another base (conversion step),
(2) Labeled deoxyguanosine 5′-three using as a template the complementary strand (that is, antisense strand) of the DNA strand containing the converted base sequence derived from the base sequence to be measured of the obtained double-stranded DNA. A step of performing a DNA amplification operation in the presence of phosphoric acid (dGTP) (amplification step); and (3) a DNA strand containing an amplified base sequence corresponding to the converted base sequence of the amplified double-stranded DNA. (Ie, sense strand) or a step of measuring the amount of labeled guanine in the amplified double-stranded DNA (analysis step)
The method for measuring the methylation rate of cytosine in a base sequence to be measured (hereinafter referred to as the antisense strand template-G type measurement method of the present invention).

  Furthermore, this invention relates to the cell or tissue which measured the methylation rate of cytosine by the said measuring method.

In the present invention, (1) the contact between the chromosomal specimen of the cell and the anti-methylated cytosine antibody is carried out under conditions that can dissociate the monovalent bond in the antigen-antibody reaction and maintain the bivalent bond. And (2) a method for evaluating a cell state, comprising a step of detecting an anti-methylated cytosine antibody bound to the chromosome specimen.

  As used herein, “cytosine” means unmethylated cytosine, that is, unmethylated cytosine. “Methylated cytosine” means 5-methylcytosine.

In the present specification, the “base sequence to be measured” means a base sequence to be measured for the DNA methylation rate by the method of the present invention. In the method of the present invention, an arbitrary base sequence on a single-stranded DNA or an arbitrary base sequence on an arbitrary DNA strand in double-stranded DNA can be used as a measurement target base sequence.
More specifically, as shown in FIG. 3, in the DNA genome, structural genes (for example, structural gene A and structural gene B in FIG. 3) are encoded in both the plus (+) strand and the minus (−) strand. ing. For example, when the double-stranded DNA shown in FIG. 3 is used as a test sample, for example, the base sequence of the structural gene A on the + strand or a partial base sequence thereof, the 5 ′ upstream region a of the structural gene A ( The nucleotide sequence of the 5 ′ upstream region b of the structural gene B (including the promoter region), or a partial nucleotide sequence thereof, or The base sequence of the complementary region b ′ of the region b or a partial base sequence thereof can be used as the measurement target base sequence.

  Further, in this specification, the “base sequence after conversion” means that the measurement base sequence is treated with a cytosine modifier capable of converting cytosine into another base (hereinafter simply referred to as cytosine modifier). Means the resulting base sequence [see the sense strand of the double-stranded DNA (b) in FIG. 1 or the sense strand of the double-stranded DNA (f) in FIG. 2].

  Further, in this specification, the “base sequence after amplification” refers to the base sequence after conversion in a double-stranded DNA obtained by DNA amplification operation using a DNA strand containing the base sequence after conversion or a complementary strand thereof as a template. Means the corresponding sequence. For example, as shown in FIG. 1, when DNA amplification operation is performed using a DNA strand containing the converted base sequence [sense strand of double-stranded DNA (b) in FIG. 1] as a template, double-stranded DNA ( c) or a sequence on the sense strand of double-stranded DNA (d). On the other hand, as shown in FIG. 2, when a DNA amplification operation is carried out using a complementary strand of the DNA strand containing the converted base sequence [the antisense strand of double-stranded DNA (f) in FIG. 2] as a template, It means the sequence on the sense strand of double-stranded DNA (g) or double-stranded DNA (h).

  In the present specification, a DNA strand containing a base sequence to be measured, a base sequence after conversion, or a base sequence after amplification in double-stranded DNA is referred to as “sense strand”, and its complementary strand is referred to as “antisense strand”. ". In general, a DNA strand containing a biologically meaningful base sequence (for example, a base sequence encoding a protein) is referred to as a sense strand, and a complementary strand thereof is referred to as an antisense strand. As specified, a DNA strand containing a base sequence to be measured, a base sequence after conversion, or a base sequence after amplification is referred to as a sense strand regardless of whether or not it contains a biologically meaningful base sequence, and its complementary strand Is referred to as the antisense strand.

  According to the measurement method of the present invention, an arbitrary region can be used as a target sequence without analyzing a methylation site in advance. In addition, the DNA methylation rate can be measured easily and accurately.

  More specifically, in the measurement method of the present invention, a cytosine methylation site may or may not be contained in a region to which a PCR primer that amplifies a region to be subjected to methylation analysis hybridizes. This means that it is not necessary to analyze in advance the methylation sites of individual genes to be analyzed, and CpG islands and promoter regions are divided from the genome information by computer analysis, and PCR is performed based on the analysis results. This means that it is possible to design primers.

Furthermore, according to the measurement method of the present invention, it is possible to measure the methylation rate of an arbitrary region sandwiched between PCR primers. Thereby, it is possible to quantitatively analyze the methylation rate of not only the region where the PCR primer hybridizes as in the conventional method but also the entire region sandwiched by the PCR primer. In the measurement method of the present invention, since a DNA amplification method (particularly, PCR amplification method) is applied, a very small amount of sample can be analyzed.
Serum and urine contain several ng / mL to several hundred ng / mL of free DNA fragments, but their quantitative change is an effective means for diagnosing cancer, especially for gallbladder. It has been reported. Since the measurement method of the present invention can also analyze a small amount of blood free DNA fragment or urine free DNA fragment derived from such a specific tissue, a plurality of genes contained in this free DNA can be analyzed. By analyzing the profile of the methylation pattern, it is possible to determine which tissue-derived DNA it is.

  Further, according to the evaluation method of the present invention, it is possible to determine the state of a cell, for example, the malignancy of a tumor cell, the differentiation state of the cell, the safety of the cell for biological transplantation, the aging of the cell, etc. Cells and abnormal cells can be distinguished.

[1] Measurement method of the present invention Measurement method of the present invention (sense strand template-C type measurement method, sense strand template-G type measurement method, antisense strand template-C type measurement method, and antisense strand template-G type Including measurement methods)
(1) a step of treating single-stranded or double-stranded DNA containing a base sequence to be measured with a cytosine modifying agent capable of converting cytosine into another base (conversion step),
(2) A step of performing a DNA amplification operation in the presence of labeled dCTP or labeled dGTP using either one of the obtained double-stranded DNA or the obtained single-stranded DNA as a template ( (Amplification step), and (3) a step of measuring the amount of labeled cytosine or labeled guanine in any one DNA strand of the amplified double-stranded DNA or in the amplified double-stranded DNA (analysis step)
including.

  In the measurement method of the present invention, in the analysis step, (A) after separating either the sense strand or the antisense strand from the double-stranded DNA amplified in the amplification step, the labeled cytosine in the DNA strand or The amount of labeled guanine can also be measured (hereinafter referred to as a double-stranded separation type measurement method), or (B) labeled cytosine or labeled guanine in the double-stranded DNA amplified in the amplification step. Can also be measured in the double-stranded state without separation (hereinafter referred to as a double-stranded non-separated measurement method).

For example, double strand separation-sense strand template-C type measurement method is
(1) conversion step,
(2) Amplification process,
(3) a step of separating a DNA strand (sense strand) containing an amplified base sequence corresponding to the converted base sequence from the amplified double-stranded DNA (hereinafter referred to as a separation step), and (4) separated A step of measuring the amount of labeled cytosine in the DNA strand (hereinafter referred to as post-separation analysis step)
including.

In addition, double-strand separation-sense strand template-G type measurement method is:
(1) conversion step,
(2) Amplification process,
(3) a step (separation step) of separating a complementary strand (that is, an antisense strand) of a DNA strand containing an amplified base sequence corresponding to the converted base sequence from the amplified double-stranded DNA; and (4) A step of measuring the amount of labeled guanine in the separated DNA strand (analysis step after separation)
including.

In addition, double-strand separation-antisense strand template-C type measurement method is:
(1) conversion step,
(2) Amplification process,
(3) a step (separation step) of separating a complementary strand (that is, an antisense strand) of a DNA strand containing an amplified base sequence corresponding to the converted base sequence from the amplified double-stranded DNA; and (4) A step of measuring the amount of labeled cytosine in the separated DNA strand (analysis step after separation)
including.

Furthermore, double-strand separation-antisense strand template-G type measurement method is:
(1) conversion step,
(2) Amplification process,
(3) a step (separation step) of separating a DNA strand (that is, sense strand) containing an amplified base sequence corresponding to the converted base sequence from the amplified double-stranded DNA (ie, a sense strand), and (4) the separated DNA strand For measuring the amount of labeled guanine in the sample (post-separation analysis step)
including.

  Since methylation of genomic DNA in higher animals often involves cytosine methylation in the continuous sequence of CG, when measuring the methylation rate with the target only on the CG sequence, or measuring the approximate methylation rate In this case, the CG sequence of the antisense strand can be used as a target for amplification. That is, when the cytosine of the CG sequence in the sense strand is methylated, the cytosine of the corresponding CG sequence in the antisense strand is immediately methylated in the cell. Therefore, when the methylation rate is measured only with the CG sequence as a target, the number of methylated cytosines in the sense strand and the number of methylated cytosines in the antisense strand are almost the same, so methylation in the antisense strand By measuring the number of cytosines, the number of methylated cytosines in the sense strand can be determined. The antisense strand template measurement method of the present invention (including the antisense strand template-C type measurement method and the antisense strand template-G type measurement method) is based on such a principle.

  In addition, when analyzing including methylation of cytosine in DNA other than the CG sequence, the methylation pattern may not be similarly methylated in the sense strand and the antisense strand (for example, the sense strand). When the cytosine of the CA sequence is methylated, the corresponding base sequence is TG and there is no cytosine in the antisense strand. In this case, the method for measuring the sense strand template of the present invention (sense strand) Template-C type measurement method and sense strand template-G type measurement method).

Hereinafter, the measurement method of the present invention will be described based on the sense strand template measurement method of the present invention (particularly sense strand template-C type measurement method), and if necessary, the antisense strand template measurement method of the present invention will also be described. explain.
Table 1 summarizes the outline of the sense strand template-C type measurement method, sense strand template-G type measurement method, antisense strand template-C type measurement method, and antisense strand template-G type measurement method of the present invention. . In Table 1, “S strand template” and “A strand template” mean “sense strand template” and “antisense strand template”, respectively. Further, “dCTP” or “dGTP” described in the “labeled dNTP” column means a labeled deoxyribonucleoside 5′-triphosphate used at least in each method, and as described later, other than this It is also possible to use labeled deoxyribonucleoside 5′-triphosphate together. In the “operation example” column, the correspondence between each method and each operation shown in FIG. 1 and FIG. 2 is described.

1 and 2 show the sense strand template-C type measurement method, sense strand template-G type measurement method, antisense strand template-C type measurement method, and antisense strand template-G type measurement method of the present invention. It is explanatory drawing which shows typically the aspect which analyzes the signal derived from a label | marker about each of after separating a double strand.
1 and 2, the symbol “m” in the double-stranded DNAs (a) and (b) and the double-stranded DNAs (e) and (f) indicates that cytosine (C) is methylated. . The symbol “*” in the double-stranded DNAs (c) and (d) and the double-stranded DNAs (g) and (h) indicates that cytosine (C) or guanine is labeled. Furthermore, a base using lowercase letters (a, c, g, t, u) as a symbol indicating a base indicates that the base has changed in each step.
Moreover, the sequence (a), the sequence (b), the sequence (c), and the sequence (d) in FIG. 1 are base sequences represented by SEQ ID NOs: 16 to 19, respectively, and the sequence (e) in FIG. The sequence (f), the sequence (g), and the sequence (h) are base sequences represented by SEQ ID NOs: 20 to 23, respectively.

<Table 1>
S chain template / C type S chain template / G type A chain template / C type A chain template / G type
Template for amplification process Sense strand Sense strand Antisense strand Antisense strand labeled dNTP dCTP dGTP dCTP dGTP
Target of analysis process Sense strand Antisense strand Antisense strand Sense strand
Operation example Fig. 1 (A), (B) Fig. 1 (A), (C) Fig. 2 (D), (E) Fig. 2 (D), (F)

  In the conversion step in the measurement method of the present invention, the DNA (sample DNA) containing the base sequence to be measured is treated with a cytosine modifying agent capable of converting cytosine (that is, unmethylated cytosine) into another base. All cytosine in the sample DNA is chemically converted to another base [see step (A) in FIG. 1 or step (D) in FIG. 2].

  As long as the sample DNA to which the measurement method of the present invention can be applied is a DNA containing the base sequence to be measured and may contain methylated cytosine (5-methylcytosine), For example, genomic DNA or a fragment thereof or a DNA construct containing them, cDNA or a fragment thereof or a DNA construct containing them, or DNA artificially synthesized by a chemical or enzymatic method, etc. be able to. In addition, the sample DNA can be single-stranded DNA or double-stranded DNA as long as it contains the base sequence to be measured.

  The cytosine modifier used in the conversion step converts all cytosine (ie, unmethylated cytosine) contained in the sample DNA into another nucleic acid constituent base other than cytosine (eg, uracil, adenine, thymine, or guanine). It is not particularly limited as long as it can be converted (for example, chemical conversion or enzymatic conversion) and does not affect the structure of methylated cytosine. For example, cytosine modifiers that can convert cytosine to uracil include, for example, bisulfite ion (eg, C. Grunau et al., Nucleic Acids Research, 2001, Vol. 29, No. 13, e65). be able to.

  When the sample DNA is treated with bisulfite ions, for example, as shown in the double-stranded DNA (b) of FIG. 1 or the double-stranded DNA (f) of FIG. All are converted to uracil, but the other bases (adenine, guanine, thymine, and methylated cytosine) are not converted and remain the original base. Naturally, conversion from cytosine to uracil also occurs in the base sequence to be measured in the sample DNA. When all cytosines are converted to uracil, there is no cytosine (ie, unmethylated cytosine) in the converted base sequence, and the bases that may be present are adenine, guanine, thymine, uracil, And methylated cytosine.

  In the amplification step in the sense strand template-C type measurement method of the present invention, when the sample DNA is a double-stranded DNA, the sense strand of the DNA obtained in the conversion step (that is, including the converted base sequence) The sense strand and its complementary strand are amplified in the presence of labeled dCTP using the strand) as a template [see step (B) in FIG. 1]. When the sample DNA is a single-stranded DNA, the single-stranded DNA obtained in the conversion step is used as a template, and the single-stranded DNA and its complementary strand are amplified in the presence of labeled dCTP. . In the amplified double-stranded DNA, for example, as shown in the double-stranded DNA (c) of FIG. 1, methylated cytosine in the converted base sequence is substituted with labeled cytosine, uracil is substituted with thymine, and adenine , Guanine, and thymine remain the original base.

  In the sense strand template-C measurement method of the present invention, the base sequence to be measured when a cytosine modifier (for example, bisulfite ion) capable of converting cytosine to uracil is used as the cytosine modifier, after conversion Table 2 shows the correspondence between bases in the base sequence and the base sequence after amplification. In addition, the symbol (*) in Table 2 indicates a labeled cytosine.

<Table 2>
Base sequence to be measured Base sequence after conversion Base sequence after amplification
Methylated cytosine Methylated cytosine Cytosine ^(*)
Cytosine uracil thymine adenine adenine adenine guanine guanine guanine
Thymine thymine thymine

The amplification step in the sense strand template-G type measurement method of the present invention is the same as the amplification step in the sense strand template-C type measurement method of the present invention, except that labeled dGTP is used instead of labeled dCTP. [See step (C) in FIG. 1]. In the amplified double-stranded DNA, for example, as shown in the double-stranded DNA (d) of FIG. 1, methylated cytosine in the converted base sequence is substituted with cytosine, guanine is substituted with labeled guanine, and uracil. Is replaced by thymine, and adenine and thymine remain the original base.
In the double-stranded DNA amplified in the amplification step, the number of cytosines in the sense strand derived from methylated cytosine is the same as the number of guanines in the antisense strand, so by measuring the number of guanines in the antisense strand, the sense strand The number of cytosines in (ie, the number of methylated cytosines from which they are derived) can be determined. The sense strand template-G type measuring method of the present invention is based on such a principle.

  When the sample DNA is a double-stranded DNA, the DNA obtained in the conversion step is also a double-stranded DNA. Since cytosine is converted to uracil, the sense strand of the DNA obtained in the conversion step and The antisense strand is no longer perfectly complementary [see, eg, double-stranded DNA (b) in FIG. 1 or double-stranded DNA (f) in FIG. 2]. In the amplification step in the sense strand template measuring method of the present invention, the methylation rate of cytosine in the measurement target base sequence can be accurately measured by amplifying only the sense strand.

  As a DNA amplification method that can be used in the amplification step in the sense strand template measurement method of the present invention, as long as it is a method that can incorporate labeled dCTP or labeled dGTP and can amplify only the sense strand, The method is not particularly limited, and examples thereof include a polymerase chain reaction (PCR) method and a DNA amplification method using a strand displacement type DNA polymerase. As a DNA amplification method using a strand displacement type DNA polymerase, for example, a DNA amplification method using Phi29 DNA polymerase derived from bacteriophage (Amersham Biosciences; Dean, F. et al., Genome Research, 11, 1095-1099, 2001). Lizardi, P. et al., Nat. Genet., 19, 225-232, 1998), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method (Takara Bio; Patent No. 3433929), Alternatively, a LAMP (Loop-mediated isothermal amplification) method (Eiken Chemical; Patent No. 3313358) may be used.

The primer used in the amplification step in the sense strand template measurement method of the present invention can be designed as appropriate depending on, for example, the type of DNA amplification method used and the preparation method of single-stranded DNA used in the separation step described later. It is.
For example, when the PCR method is used as the DNA amplification method, a DNA region consisting of the converted base sequence of the DNA sense strand (that is, the strand containing the converted base sequence) obtained in the conversion step is double-stranded. Primer sets that can be amplified as DNA can be designed and used. Specifically, a reverse primer comprising a base sequence complementary to the 3 ′ downstream base sequence (usually 18 to 40 bases) immediately following the base sequence after conversion of the DNA sense strand obtained in the conversion step, A combination with a forward primer comprising a base sequence upstream of the 5 ′ side of the base sequence after conversion (usually 18 to 40 bases) can be used.

  The primer set used in the amplification step in the sense strand template measurement method needs to be a primer set that does not amplify the DNA antisense strand obtained in the conversion step. For example, when the forward primer is a DNA antisense strand obtained in the conversion step and a primer that does not anneal under the PCR reaction conditions, the conversion is performed without amplifying the DNA antisense strand obtained in the conversion step. Only the DNA sense strand obtained in the process can be amplified.

  As the forward primer or reverse primer used in the amplification step, one kind of primer is usually used, but whether the cytosine is included in the target base sequence to which the primer hybridizes and whether or not the cytosine is methylated. When it is not clear, it is preferable to use a mixture of two or more kinds of primers as the forward primer (or reverse primer). Alternatively, instead of using a mixture of two or more kinds of primers, it is preferable to use a primer containing a universal base capable of binding to a plurality of bases as a base corresponding to cytosine whose presence or absence of methylation is unknown.

  When cytosine is included in the target base sequence to which the primer hybridizes and it is not clear whether the cytosine is methylated or not, this cytosine (hereinafter referred to as unknown cytosine) is treated with a cytosine modifying agent to give uracil. (When the unknown cytosine is unmethylated cytosine) and when it remains methylated cytosine (when the unknown cytosine is methylated cytosine) [for example, double-stranded DNA in FIG. See (a) and (b)]. Therefore, for example, when designing a reverse primer, a primer corresponding to the unknown cytosine is adenine and a primer that is guanine, and a mixture thereof is used to amplify DNA in any case. Can be carried out. On the other hand, when designing a forward primer, a primer whose base corresponding to an unknown cytosine is thymine and a primer that is cytosine are designed and a mixture thereof is used to perform DNA amplification in any case. It becomes possible.

  When a mixture of two or more kinds of primers is used, at least one or more different bases are included, so that when the base lengths of the primers are equal, the Tm of each primer may differ. When primers having different Tm are used at the same time, DNAs derived from a specific primer tend to be preferentially amplified because the binding power to the template DNA is different. Therefore, in order to avoid such a bias, it is preferable to match the Tm by adjusting the base length of the primer.

  On the other hand, when a primer including a universal base capable of binding to a plurality of bases is used instead of a mixture of two or more kinds of primers, an appropriate universal base can be selected according to the target base. For example, as described above, when designing a reverse primer for a target base sequence containing an unknown cytosine, instead of designing a primer whose base corresponding to the unknown cytosine is adenine and a primer that is guanine, A primer in which the base corresponding to the unknown cytosine is a universal base capable of binding to both thymine and cytosine [for example, 2-amino-6-methoxyaminopurine] may be designed. .

Various bases are known as such universal bases [PK Lin and DM Brown, Nucleic Acids Research, vol. 20, No. 19, pp5149-5152, 1992]. Universal bases capable of binding to both adenine and guanine include, for example, 6H, 8H-3,4-dihydropyrimido [4,5-c] [1,2] oxazin-7-one (6H, 8H-3 , 4-dihydropyrimido [4,5-c] [1,2] oxazin-7-one) can be used. In addition, deoxyinosine is widely used as a compound that does not select a corresponding base [E. Ohtsuka et al., J. Biological Chemistry, vol. 260, No. 5, pp2605-2608, 1985].
Further preferred embodiments of the primers used in the amplification step will be further described in detail when explaining the method for preparing single-stranded DNA in the separation step.

  The labeled substance used for labeling labeled dCTP or labeled dGTP used in the amplification step in the sense strand template measurement method of the present invention does not suppress or inhibit the DNA amplification reaction in the amplification step, or suppresses or inhibits it. The labeling substance itself emits a signal (for example, fluorescence, luminescence, or isotope), or there is a partner that can specifically bind to the labeling substance. For example, the fluorescent substances FITC (Fluorescein isothiocyanate), Cy3 (Cyanine 3), Cy5 (Cyanine 5), rhodamine, Texas Red, HEX (Hexachloro-6-carboxyfluorescein), FAM (6-Carboxyfluorescein), TET (Tetrachloro-6-carboxyfluorescein), TAMRA (6-Carboxytetramethylrhodamine), TRITC (Tetramethylrhodamin), R OX (Carboxy-X-rhodamine), DNP (Dinitrophenol), digoxigenin, biotin, Alexa Fluor (Molecular Probes), etc. can be mentioned. In addition, as labeled dCTP or labeled dGTP, for example, 5-methyl dCTP, 6-methoxy dGTP, 7-methyl dGTP, or the like can be used.

DNA amplification in the amplification step in the sense strand template measurement method of the present invention is performed in the presence of labeled dCTP or labeled dGTP using the double-stranded DNA sense strand or single-stranded DNA obtained in the conversion step as a template. Except for this, it can be carried out according to the DNA amplification method used.
For example, when the PCR method is used as a DNA amplification method in the sense strand template-C type measurement method, in addition to labeled dCTP, dATP, dGTP, and dTTP, and optionally dCTP (ie, unlabeled dCTP) In addition, PCR is performed. In the measurement method of the present invention, only labeled dCTP can be used as dCTP, or a mixture of labeled dCTP and unlabeled dCTP can be used. When a mixture of labeled dCTP and unlabeled dCTP is used, for example, by drawing a standard curve using methylated standard DNA in which 5-methyl dCTP is incorporated in an arbitrary amount, labeled nucleotides can be reacted in a PCR reaction. Even if only a very small amount is added to the solution, the actual methylation rate of the sample DNA for analyzing the methylation rate can be calculated from the calibration curve. For example, it has been confirmed that the amount of fluorescently labeled nucleotide is actually measurable with an unlabeled: label ratio of 500: 1 or less.
When PCR is used as a DNA amplification method in the sense strand template-G measurement method, in addition to labeled dGTP, dATP, dCTP, and dTTP, and optionally dGTP (ie, unlabeled dGTP) are added. In addition, PCR is performed.

  In addition, when the PCR method is used as the DNA amplification method, an asymmetric PCR method can also be used. In the asymmetric PCR method, for example, the sense strand can be preferentially amplified by performing PCR using a reverse primer and a forward primer in a quantitative ratio of about 1:50.

In the amplification step in the sense strand template-C measurement method, in addition to the labeled dCTP, in place of (or in addition to) at least one deoxyribonucleoside 5′-triphosphate of dATP, dGTP, or dTTP, the labeled dCTP It is possible to use deoxyribonucleoside 5′-triphosphate (eg, labeled dATP, labeled dGTP, or labeled dTTP) labeled with a labeling substance different from the labeling substance used for labeling. If the sample DNA is the same gene, the numbers of adenine and guanine in the sense strand and cytosine and thymine in the antisense strand in each sample DNA are not changed. Therefore, the methylation rate can be accurately quantified by quantifying methylated cytosine using adenine, guanine, or thymine as an internal standard.
In the amplification step in the sense strand template-G type measurement method, in addition to (or in addition to) the labeled dGTP, at least one deoxyribonucleoside 5′-triphosphate of dATP, dCTP, or dTTP is used instead of (or in addition) a label. Deoxyribonucleoside 5′-triphosphate (for example, labeled dATP, labeled dCTP, or labeled dTTP) labeled with a labeling substance different from the labeling substance used for labeling the labeled dGTP can be used.

  In addition, as a forward primer used in the amplification step, a forward primer labeled with a labeling substance different from the labeling substance used for labeling labeled dCTP or labeled dGTP can also be used. For example, when the first fluorescent substance (fluorescent substance A) is used for labeling of the labeled forward primer and the second fluorescent substance (fluorescent substance B) is used for labeling the labeled dCTP, an analysis step described later , The fluorescence intensity of the sense strand DNA is measured at the respective measurement wavelengths of the fluorescent substances A and B, and the value of (fluorescence intensity of the fluorescent substance B) / (fluorescence intensity of the fluorescent substance A) is determined in the sense strand DNA. The percentage of cytosine contained can be calculated.

  As described above, the amplification step in the measurement method of the present invention has been described based on the sense strand template measurement method of the present invention. However, the amplification step in the antisense strand template measurement method of the present invention also includes the amplification step of the DNA obtained in the conversion step. It can be carried out in the same manner as the amplification step in the sense strand template measurement method of the present invention except that the antisense strand (that is, the complementary strand of the DNA strand containing the converted base sequence) is used as a template [FIG. Step (E) or (F)].

  Specifically, in the amplification step in the antisense strand template-C measurement method of the present invention, the antisense strand is prepared by using the antisense strand of the DNA obtained in the conversion step as a template and in the presence of labeled dCTP. And its complementary strand is amplified [step (E) in FIG. 2]. In the amplified double-stranded DNA, for example, as shown in the double-stranded DNA (g) of FIG. 2, methylated cytosine is replaced with labeled cytosine, uracil is replaced with thymine, and adenine, guanine, and thymine are The original base remains.

Further, in the amplification step in the antisense strand template-G type measurement method of the present invention, the antisense strand and its complement in the presence of labeled dGTP using the antisense strand of the DNA obtained in the conversion step as a template. Amplify the strand [see step (F) in FIG. 2]. In the amplified double-stranded DNA, for example, as shown in the double-stranded DNA (h) of FIG. 2, methylated cytosine in the converted base sequence is replaced with cytosine, guanine is replaced with labeled guanine, and uracil. Is replaced by thymine, and adenine and thymine remain the original base.
As the labeled substance used in the labeling of labeled dCTP or labeled dGTP in the amplification step in the antisense strand template measurement method of the present invention, for example, the labeling substance exemplified in the amplification step in the C-type measurement method of the present invention is used. be able to.

As a DNA amplification method that can be used in the amplification step in the antisense strand template measurement method of the present invention, labeled dCTP or labeled dGTP can be incorporated, and the antisense strand (that is, the converted nucleotide sequence is converted into a nucleotide sequence). The method is not particularly limited as long as it is a method capable of amplifying only the complementary strand), and for example, the DNA amplification method exemplified in the amplification step in the sense strand template measurement method of the present invention can be used.
When the PCR method is used as the DNA amplification method in the antisense strand template measurement method of the present invention, the conversion of the DNA antisense strand obtained in the conversion step (that is, the complementary strand of the strand containing the base sequence after conversion) A primer set capable of amplifying a DNA region corresponding to the post-base sequence as a double-stranded DNA can be designed and used. In this case, the primer set needs to be a primer set that does not amplify the DNA sense strand obtained in the conversion step. For example, if the reverse primer is a DNA sense strand obtained in the conversion step and a primer that does not anneal under the PCR reaction conditions, the reverse primer can be obtained in the conversion step without amplifying the DNA sense strand obtained in the conversion step. Only the resulting DNA antisense strand can be amplified.

When the PCR method is used as the DNA amplification method in the antisense strand template-C measurement method of the present invention, in addition to labeled dCTP, dATP, dGTP, and dTTP, and optionally dCTP (ie, unlabeled dCTP) In addition, PCR can be performed. When the PCR method is used as the DNA amplification method in the antisense strand template-G type measurement method of the present invention, in addition to labeled dGTP, dATP, dCTP, and dTTP, and optionally dGTP (ie, unlabeled) (dGTP) and PCR can be performed.
In addition, when the PCR method is used as the DNA amplification method in the antisense strand template measurement method of the present invention, an asymmetric PCR method can also be used. In the asymmetric PCR method, for example, the antisense strand can be preferentially amplified by performing PCR using a forward primer and a reverse primer in a quantitative ratio of about 1:50.

  In the amplification step in the antisense strand template-C measurement method of the present invention, in addition to (or in addition to) the labeled dCTP, at least one deoxyribonucleoside 5′-triphosphate of dATP, dGTP, or dTTP. Deoxyribonucleoside 5′-triphosphate (for example, labeled dATP, labeled dGTP, or labeled dTTP) labeled with a labeling substance different from the labeling substance used for labeling of labeled dCTP may be used. it can. Further, in the amplification step in the antisense strand template-G type measurement method of the present invention, in addition to (or in addition to) the labeled dGTP, at least one deoxyribonucleoside 5′-triphosphate of dATP, dCTP, or dTTP. And deoxyribonucleoside 5′-triphosphate (eg, labeled dATP, labeled dCTP, or labeled dTTP) labeled with a labeling substance different from the labeling substance used for labeling of labeled dGTP. be able to.

  Further, as one primer used in the amplification step, a reverse primer labeled with a labeling substance different from the labeling substance used for labeling labeled dCTP or labeled dGTP can also be used. For example, when the first fluorescent substance (fluorescent substance A) is used for labeling the labeled reverse primer and the second fluorescent substance (fluorescent substance B) is used for labeling the labeled dCTP, an analysis step described later , The fluorescence intensity of the antisense strand DNA is measured at each measurement wavelength of the fluorescent substances A and B, and the value of (fluorescence intensity of the fluorescent substance B) / (fluorescence intensity of the fluorescent substance A) is calculated as the antisense strand DNA. The ratio of cytosine contained therein can be calculated.

In the separation step in the double-strand separation measurement method of the present invention, a sense strand (in the case of double-strand separation-sense strand template measurement method) or antisense strand (two strands) is obtained from the double-stranded DNA amplified in the amplification step. Strand separation-in the case of the antisense strand template measurement method).
The DNA obtained in the amplification step is usually a double-stranded DNA, and both the sense strand and the antisense strand are labeled dCTP (in the case of double-strand separation-C type measurement method) or labeled dGTP ( In the case of double-strand separation-G type measurement method) or labeled dCTP and labeled dGTP (when two types of labeled deoxyribonucleoside 5′-triphosphate are used) have been introduced. In the double-strand separation type measuring method of the present invention, since the double-stranded DNA is separated into the sense strand and the antisense strand, when measuring the amount of the labeling substance on one DNA strand, the labeling substance on the other DNA strand Will not be affected. Hereinafter, the separation step in the double-strand separation measurement method of the present invention will be described by taking the case where the sense strand is separated as an example. However, those skilled in the art can similarly perform the same by making obvious modifications as necessary. The antisense strand can be separated.

As a single-stranded DNA preparation method for separating only the sense strand, for example,
(1) In the state of double-stranded DNA, after immobilizing only the sense strand on the immobilization carrier, the double-stranded DNA is denatured, so that only the antisense strand not immobilized on the immobilization carrier is obtained. Dissociation and removal method (hereinafter referred to as immobilization method),
(2) a method in which only the antisense strand is degraded with an enzyme (hereinafter referred to as an enzymatic degradation method),
(3) Separation method using molecular sieving effect (for example, electrophoresis) (hereinafter referred to as molecular sieving method) or (4) Separation method using affinity (hereinafter referred to as affinity method) )
Etc.

  The immobilization method (1) is performed, for example, by using a forward primer into which an appropriate reactive functional group has been introduced as a forward primer (forming the 5 ′ end of the sense strand) used in PCR performed in the amplification step. [In the case of separating only the antisense strand, an appropriate reactive functional group was introduced as a reverse primer (forming the 5 ′ end of the antisense strand) used in PCR performed in the amplification step] Use reverse primer]. The reactive functional group reacts with the reactive functional group on the immobilization carrier side to immobilize double-stranded DNA on the immobilization carrier, and suppresses or inhibits PCR in the amplification process. Further, there is no particular limitation as long as it is resistant to denaturation treatment (for example, acid treatment or alkali treatment) of double-stranded DNA. Examples of the reactive functional group include an amino group, a maleimide group, an aldehyde group, a carboxyl group, a mercapto group, a vinyl sulfone group, and a methanesulfonyl group. Further, in place of the reactive functional group, a combination of binding partners capable of binding to each other by a specific binding reaction can be used. As such a combination, for example, a biotin / avidin combination can be mentioned. .

  In the immobilization method, for example, double-stranded DNA is immobilized on an immobilization carrier via a reactive functional group or a binding partner introduced into a forward primer. In this case, of the double-stranded DNA, the sense strand is immobilized on the immobilization carrier, while the antisense strand is hydrogen bonded to the sense strand, but is immobilized on the immobilization carrier by a covalent bond. It has not become. In this state, when a double-stranded DNA denaturation treatment [for example, acid treatment, alkali treatment, heat treatment, or DNA denaturant treatment (eg, formamide, dimethylformamide, dimethylsulfoxide, etc.) treatment] And the antisense strand dissociate, so that only the antisense strand that is not immobilized on the immobilization carrier is detached from the immobilization carrier. Single strand DNA can be prepared by removing the detached antisense strand by, for example, washing.

Examples of the enzymatic degradation method (2) include a method using an exonuclease (for example, λ exonuclease) that degrades from the end of double-stranded DNA in the 5 ′ to 3 ′ direction.
In this method, for example, as a primer set used for PCR performed in an amplification step, a reverse primer that easily digests to the exonuclease (forms the 5 ′ end of the antisense strand) and a sense primer that hardly digests to the exonuclease ( (The 5 ′ end of the sense strand is formed) and a combination with the above-mentioned [in the case of separating only the antisense strand, the exo is used as a primer set used in PCR performed in the amplification step. Use a combination of a forward primer that is easily digested by nuclease (forms the 5 ′ end of the sense strand) and a reverse primer that is difficult to digest by the exonuclease (forms the 5 ′ end of the antisense strand)]. Examples of a method for making a primer easily digestible with exonuclease include a method for phosphorylating the 5 ′ end of the primer. Further, as a method for making the primer exonuclease indigestible, for example, a method in which the 5 ′ end of the primer is left as a hydroxyl group (—OH), or at least one (preferably all) internucleotide bond is used. Examples of the method include phosphorothioate-type linkage, and combining them can further improve exonuclease indigestibility.
Further, the reactive functional group or binding partner described in the immobilization method can be further introduced into the exonuclease indigestible forward primer.

In the enzymatic degradation method using the exonuclease, for example, (a) double-stranded DNA is immobilized on an immobilization carrier in advance, the double-stranded DNA is treated with exonuclease, and then digested products are removed by washing. Can be used to prepare single-stranded DNA, or (b) the double-stranded DNA is treated with exonuclease, and then the single-stranded DNA and the digested product are separated by an ordinary DNA purification method. Thus, single-stranded DNA can also be purified.
In the method (a), an exonuclease indigestible forward primer into which a reactive functional group or a binding partner is further introduced is used.
In the method (b), examples of the DNA purification method include an ethanol precipitation method, an ultrafiltration method, and a silica adsorption method.

  In the molecular sieving method (3), various separation methods using the molecular sieving effect, such as electrophoresis (for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis), gel filtration, An ultrafiltration method or a centrifugal separation method can be used. The molecular sieving method (3) or affinity method (4) can be used, for example, when an asymmetric PCR method is used in the amplification step. In electrophoresis (especially SSCP electrophoresis), for example, the PCR reaction solution obtained in the amplification step is electrophoresed on an electrophoresis gel (for example, agarose gel or polyacrylamide gel) by a conventional method, It can be separated as a single-stranded DNA having a low migration degree in a gel. Alternatively, after performing a normal DNA amplification method (that is, not asymmetric), electrophoresis is performed immediately after denaturation of double-stranded DNA, so that each single-stranded DNA having a different base sequence or composition can be obtained in a gel. Can be separated. Further, even when a denaturing agent is mixed in the gel or a denaturing agent concentration gradient is formed in the gel, each single-stranded DNA can be similarly separated.

  In the molecular sieving method, the physical or chemical properties of each DNA strand can be changed by fluorescent substance labeling to separate the DNA strands according to differences in physical properties. For example, double-stranded DNA can be separated by increasing the hydrophobicity of one DNA strand or increasing the ionicity of one DNA strand by fluorescent substance labeling.

In the analysis step in the measurement method of the present invention, in the case of the double-stranded separation type measurement method, the amount of labeled deoxyribonucleoside 5′-triphosphate in one of the DNA strands obtained in the separation step is measured. Thus, the methylation rate of cytosine [or the ratio of cytosine (ie, unmethylated cytosine) to methylated cytosine] in the base sequence to be measured is determined. In each embodiment, the DNA strand to be analyzed and the labeled deoxyribonucleoside 5′-triphosphate are as shown in Table 1 above.
That is, in the double-strand separation-sense strand template-C type measurement method, the amount of labeled cytosine in the sense strand [see double-stranded DNA (c) in FIG. 1], double-strand separation-sense strand template-G In the type measurement method, the amount of labeled guanine in the antisense strand [see double-stranded DNA (d) in FIG. 1], double-strand separation-antisense strand template-in the C-type measurement method, label in the antisense strand In the double strand separation-antisense strand template-G type measurement method, the amount of labeled guanine in the sense strand is determined according to the double-stranded DNA (g) in FIG. Each strand DNA (h)] is measured.

  For example, in the sense strand template-C type measurement method, as also shown in Table 2 above, cytosine in the base sequence after amplification corresponds to methylated cytosine in the base sequence to be measured. Therefore, in the sense strand template-C type measurement method, the amount of methylated cytosine in the measurement target base sequence can be calculated by measuring the amount of cytosine (ie, labeled cytosine) in the base sequence after amplification. it can.

On the other hand, in the case of a double-stranded non-separation type measurement method, the base sequence to be measured is determined by measuring the amount of labeled deoxyribonucleoside 5′-triphosphate in the double-stranded DNA obtained in the amplification step. The cytosine methylation rate [or the ratio of cytosine (ie, unmethylated cytosine) to methylated cytosine] is determined.
That is, in the double-stranded non-separated-sense strand template-C type measurement method or the double-stranded non-separated-antisense strand template-C type measurement method, the amount of labeled cytosine in the double-stranded DNA In the separated-sense strand template-G type measurement method or the double-stranded non-separated-antisense strand template-G type measurement method, the amount of labeled guanine in the double-stranded DNA is measured.

The amount of labeled cytosine or labeled guanine can be measured by a conventional method according to the type of labeling substance used for labeling. Hereinafter, although an analysis process is demonstrated based on the aspect at the time of using labeled cytosine, it can implement similarly also when using labeled guanine.
For example, when the labeling substance itself emits a signal (for example, fluorescence, luminescence, or isotope) or is specifically identified using an isotope, for example, use a measuring device according to the signal. Thus, the amount of the labeling substance (that is, the amount of labeled cytosine) can be measured.
When there is a partner that can specifically bind to the labeling substance, for example, the partner is labeled with another labeling substance (for example, a fluorescent substance, a luminescent substance, an isotope, or an enzyme), and this labeling is performed. After binding the partner to labeled cytosine, the amount of labeled cytosine can be determined by measuring the signal of the labeled partner. Alternatively, a method capable of measuring an extremely small change in mass without labeling the partner [for example, a measurement method using surface plasmon resonance (SPR) or a measurement method using crystal oscillator microbalance (CM)] is used. By using it, the binding amount of the partner to the labeling substance can be directly measured, and the amount of labeled cytosine can be determined. Furthermore, the labeling substance of the partner can be obtained by measuring the molecular weight after binding the partner (for example, by determining the molecular weight by electrophoresis in electrophoresis or using a mass spectrometer) without labeling the partner. The amount of binding to can be calculated and the amount of labeled cytosine can be determined.

  In the measurement method of the present invention, the conversion step, the amplification step, and the analysis step are performed using only the DNA to be measured, and the labeled nucleotide in the double-stranded DNA derived from the DNA to be measured or one of the DNA strands The methylation rate in the measurement target base sequence of the measurement target DNA can also be determined by directly measuring the amount of uptake of DNA, but a methylated standard DNA corresponding to the measurement target DNA is prepared in advance, and the measurement target The DNA to be measured is prepared by performing a conversion step, an amplification step, and an analysis step under the same conditions using the DNA and the methylated standard DNA, and then creating a calibration curve from the analysis result of the methylated standard DNA. The methylation rate in the base sequence to be measured can also be determined.

  The measurement method of the present invention using a methylated standard DNA can be performed, for example, by the following procedure. That is, by using a DNA to be measured as a template and using a primer set used in the amplification step, PCR is performed in the presence of 5-methyl dCTP alone or in the presence of 5-methyl dCTP and dCTP, whereby methylated standard DNA To prepare. In this case, a plurality of methylated standard DNAs having different methylation rates are prepared by changing the mixing ratio of 5-methyl dCTP and dCTP. For example, the mixing ratio of 5-methyl dCTP and dCTP (5-methyl dCTP: dCTP) is set to 6 levels of 0:10, 2: 8, 4: 6, 6: 4, 8: 2, and 10: 0. By carrying out PCR, six types of methylated standard DNAs with cytosine methylation rates of approximately 0%, 20%, 40%, 60%, 80%, and 100% can be prepared (note that The mixing ratio can be changed as appropriate). When determining the exact methylation rate in each methylated standard DNA, for example, with respect to each methylated standard DNA, the necessary number of base sequences before and after bisulfite ion treatment are directly sequenced, It can be determined by performing statistical processing.

  Using the obtained plurality of methylated standard DNAs and the DNA to be measured, a conversion step, an amplification step, and an analysis step are performed under the same conditions. After preparing a calibration curve from each amount of labeled nucleotide incorporation obtained for each methylated standard DNA and each methylation rate of each methylated standard DNA, the amount of labeled nucleotide incorporation obtained for the DNA to be measured Is applied to the calibration curve, the methylation rate in the measurement target base sequence of the measurement target DNA can be determined.

Although the measurement method of the present invention has been described above, the measurement method of the present invention will be further described based on a more specific operation procedure.
(1) Measuring method using molecular sieve by electrophoresis In this measuring method, the analysis after the conversion step is quantified by molecular sieve.
After the sample DNA is treated with bisulfite ions, PCR is performed using dATP, dGTP, dTTP, and biotin-labeled dCTP to incorporate biotin-labeled dCTP into the DNA product. The two types of oligo DNA primers used for the PCR are designed as follows. The 5 ′ end of one oligo DNA primer is phosphorylated to make it easily digestible to λ exonuclease. The other oligo DNA primer has a hydroxyl group (—OH) at the 5 ′ end in order to make it indigestible to λ exonuclease, or each nucleotide in order to further enhance the indigestibility. S-oligolated DNA primers in which all phosphate bonds in between or a part of phosphate bonds are converted to thiol bonds are used.
The obtained PCR product is digested with λ exonuclease to make a single strand. The avidin solution is mixed in the solution after the λ exonuclease digestion operation to prepare a sample for electrophoresis and subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gel after electrophoresis is stained with a DNA stain or a protein stain, and the molecular weight of the electrophoresis is measured. In this case, it is important that the biotin-avidin bond does not leave. In normal SDS-PAGE, the biotin-avidin bond may be separated. For example, it is preferable to reduce the SDS concentration in the sample buffer to about 0.1% under non-reducing conditions, and the SDS concentration in the gel. Is preferably 0.1% or less.

  When biotinylated cytosine is contained in the single-stranded DNA obtained by the PCR reaction, the number of avidin molecules according to the number of biotin on the single-stranded DNA is bound, and the molecular weight of the single-stranded DNA Increase. Thereby, the number of biotinylated cytosines on the single-stranded DNA can be quantified. Since avidin has four biotin binding sites per molecule, one molecule of avidin is used for 2 to 4 molecules of biotin on one single-stranded DNA when an insufficient concentration of avidin solution is used. Will not be able to accurately quantify the number of biotin molecules on the single-stranded DNA. Therefore, it is necessary to add a sufficient amount of avidin.

(2) Measurement method using fluorescence-labeled avidin or enzyme-labeled avidin PCR is carried out using a side chain for immobilization to a substrate, for example, an amino group, into either one of two oligo DNA primers. Perform amplification. Double-stranded DNA, which is a PCR product, is immobilized in a well of a maleic anhydride activated polystyrene plate [Reacti-Bind ™ Maleic Anhydride Activated Polystyrene Plates; manufactured by PIERCE, Cat # 15100] according to the product manual. After immobilization, 200 μL of blocking solution [1 mol / L glycine / 1 mmol / L-EDTA / 0.1% Tween 20 / phosphate buffered saline (PBS)] is placed in the well and left at room temperature for 1 hour or longer. Next, 100 μL of 0.3 mol / L sodium hydroxide solution is added to the well and left for 20 minutes to make a single strand of DNA. Next, the wells are washed several times with 0.1% Tween 20 / PBS, and 5 μg / mL alkaline phosphatase-labeled streptavidin-containing 0.1% Tween 20 / PBS is put into the wells and left at room temperature for 1 hour. Next, the liquid in the well is removed and washed 5 times with 0.1% Tween20 / PBS. Next, an alkaline phosphatase substrate solution (SIGMA, Cat # N-1891) is prepared according to the product manual, placed in a well, allowed to stand at room temperature for several minutes to 1 hour, and the absorbance at 405 nm is measured.

  In the above method, DNA is single-stranded in the well, but DNA that has been single-stranded by enzymatic degradation of only the DNA strand not to be measured by λ exonuclease is immobilized in the well in the same manner as described above. However, the same effect can be obtained. The oligo DNA primer for PCR used in this case is designed according to the method described above, and can decompose and remove only single-stranded DNA that is not the subject of analysis. The single-stranded DNA to be analyzed is λ exonuclease. It must be of a structure that is resistant to and yet be able to be immobilized on a substrate.

(3) Measurement method by asymmetric PCR method A PCR reaction is carried out using a forward primer labeled with fluorescent substance A out of two oligo DNA primers and further admixed with dCTP labeled with fluorescent substance B. In the PCR reaction, the reverse primer is about 1/50 of the forward primer in the reaction solution. The PCR reaction solution is electrophoresed on an agarose gel or polyacrylamide gel according to a conventional method. By the above method, the PCR product having the base sequence of the fluorescently labeled forward primer appears as a single-stranded DNA having a low mobility in the electrophoresis gel. Using a fluorescence image analyzer (manufactured by Takara Bio Inc .; FMBIO), the fluorescence intensity of the single-stranded DNA band is measured at each measurement wavelength of the fluorescent substances A and B. The ratio of cytosine contained in the single-stranded DNA can be calculated from the value of (fluorescent intensity of fluorescent substance B) / (fluorescent intensity of fluorescent substance A) in the single-stranded DNA.

(4) Measurement Method Using Surface Plasmon Resonance (SPR) or Quartz Crystal Microbalance (CM) The two methods described are basically methods for measuring a very small change in mass on a sensor chip. For example, when a substance B having affinity is bound to the substance A bound on the sensor chip, the mass on the sensor increases due to the binding, and the change can be detected. Examples of measuring instruments that use SPR include Biacore system (Biacore 2000; Biacore), and examples of measuring instruments that use CM include a quartz oscillator biosensor (AFFINIX Q4; Initiative). Can be mentioned.

  The single-stranded DNA prepared by any of the methods described above is immobilized on a substrate. At this time, cytosine in the single-stranded DNA is hapten-labeled. In addition, an amino group is previously introduced into the oligo DNA primer to be incorporated 5 'upstream of the sense strand of the DNA amplified in the PCR reaction. For example, dCTP labeled with biotin as a hapten is incorporated into a PCR product, and the single-stranded DNA is immobilized on a sensor. The immobilization of the single-stranded DNA on the sensor chip is performed via an amino group previously introduced into the DNA. By contacting the avidin solution with the sensor chip on which the single-stranded DNA to be analyzed is immobilized as described above, the avidin is contained in the single-stranded DNA immobilized on the sensor chip. The number of molecules in proportion to the amount of biotin incorporated into is bound. By measuring this mass change, it is possible to analyze how many cytosines are contained in the single-stranded DNA. In addition, when the methylation rate is compared between samples, or when methylated standard DNA is prepared and the operation is performed in the same manner as the sample, a calibration curve can be created to quantify the methylation rate.

(5) Analysis of methylation rate in the vicinity of the transcription start site of human E-cadherin Hereinafter, the measurement method of the present invention will be described using a specific sequence as an example.
Actually, in the methylation of the human genome, cytosine in a continuous sequence of -CG- is methylated, and normally, the methylated cytosine is methylated on both the sense strand and the antisense strand. For example, the following double-stranded DNA (SEQ ID NO: 1) will be described as an example. Note that cytosine surrounded by a box represents methylated cytosine.

  In the above sequence, there are two 5′-CG-3 ′ sequences in each of the sense strand and the antisense strand. Here, one of the 5′-CG-3 ′ sequences is methylated in each DNA strand. Assuming that Usually, in higher animals, when cytosine in a specific 5′-CG-3 ′ sequence having a sense strand is methylated, the 5′- of the antisense strand that is complementary to the region is complementary. Cytosine in the CG-3 ′ sequence is similarly methylated. Therefore, when measuring the methylation rate of DNA in a specific region, the sense strand may be the subject of analysis. Similarly, when the antisense strand is also the subject of analysis, the DNA methylation rate in a specific region is The same result. In addition, although the number of bases of cytosine is different between the sense strand and the antisense strand, in the analysis of the methylation rate in the present invention, in the genomic DNA of higher animals, the methyl of the region to be analyzed is analyzed regardless of which DNA strand is to be analyzed. The same analysis results in terms of conversion rate. In higher animals, cytosine present in the 5′-CG-3 ′ continuous sequence is a target for methylation. Therefore, if the methylation rate of DNA in a certain region is analyzed, the sense strand Even if the PCR is amplified by PCR, the same result can be obtained even if the antisense strand is similarly amplified and the methylation rate is analyzed. However, in higher animals, cytosine other than cytosine in the continuous sequence of CG is also subjected to methylation, so when it is to be analyzed including such methylation of cytosine, the sense strand (gene It is necessary to select the sense strand (based on the direction of transcription) as an analysis target. Specifically, when cytosine in the 5′-CA-3 ′ continuous sequence of the sense strand is methylated, the sequence of the corresponding antisense strand is 5′-TG-3 ′. , Cytosine is not included.

When only cytosine shown in the above sequence is methylated, when this is treated with bisulfite ion, the following base sequence (SEQ ID NO: 2) is obtained, and the sense strand and the antisense strand can no longer be complementarily bound. I can't.

For example, when the sense strand is to be subjected to PCR amplification, the oligo DNA primer 1 that first hybridizes complementarily to the sense strand and the first strand synthesized by the oligo DNA primer are complementary. It is necessary to design an oligo DNA primer 2 that binds automatically. In the general PCR method, there is a template DNA strand that hybridizes with two oligo DNA primers from the first cycle. However, when PCR is performed using DNA treated with bisulfite ion as a template, In the first cycle of the PCR reaction, only one oligo DNA primer is hybridized.
Because of this principle, when performing PCR using DNA treated with bisulfite ions as a template, first select which DNA strand whose base sequence was changed by bisulfite treatment was amplified. Carefully design oligo DNA primers for PCR reactions.

For example, here, it is assumed that the methylation rate of genomic DNA (SEQ ID NO: 3) of human E-cadherin in the region shown below is measured.
Base sequence before treatment with bisulfite ion (SEQ ID NO: 3):

An example of experimental conditions for PCR amplification using the human genomic DNA treated with bisulfite ion as a template DNA is shown below:
Template DNA: 1 μg of human genomic DNA treated with bisulfite ion
Forward primer: 5'tttAGTAATTTTAGGTTAGAGGGTTAT-3 '(SEQ ID NO: 6) 50pmole
["T" on the 5 'side indicates S-oligolation]
Reverse primer: 5'-AAACTCACAAATACTTTACAATTCC-3 '(SEQ ID NO: 7) 50pmole
[The 5 'end is phosphorylated]
Taq DNA polymerase: TaKaRa Taq Hot Start Version (manufactured by Takara Bio Inc.), 2.5 unit nucleotides: 2.5 μmole each of dATP, dGTP, and dTTP
Cytosine: dCTP and Texas Red labeling Add so that the total amount of dCTP added is 2.5 μmole
Bring the total volume of the PCR reaction system to 50 μL.
PCR reaction conditions:
(95 ° C, 2 minutes) x 1 cycle (95 ° C, 30 seconds / 59 ° C, 30 seconds / 72 ° C, 30 seconds) x 30 cycles (72 ° C, 8 minutes) x 1 cycle

ＰＣＲ反応に使用する２種類のオリゴＤＮＡプライマーは、上記配列（配列番号４）中の四角で囲んだ部分の塩基配列を標的とし、前記ＰＣＲ反応によって増幅されるＤＮＡ産物の塩基配列は下記配列（配列番号５）のようになる。なお、四角で囲んだ塩基は、ＰＣＲ反応で使用したオリゴＤＮＡプライマーの塩基配列を反映した領域である。
ＰＣＲ増幅後の塩基配列（配列番号５）：

Base sequence after bisulfite ion treatment (when DNA before treatment is not methylated at all: SEQ ID NO: 4):

The two types of oligo DNA primers used for the PCR reaction target the base sequence of the portion enclosed by the square in the above sequence (SEQ ID NO: 4), and the base sequence of the DNA product amplified by the PCR reaction is the following sequence ( It becomes like sequence number 5). The base surrounded by a square is a region reflecting the base sequence of the oligo DNA primer used in the PCR reaction.
Base sequence after PCR amplification (SEQ ID NO: 5):

(6) Method for preparing methylated standard DNA For the quantification of genomic DNA methylation, DNA having any specific methylation rate (methylated standard DNA) is prepared, and this is used as a reference DNA for the methylation rate. Can be used as a calibration curve to calculate the methylation rate of the sample DNA. In the PCR reaction, a DNA product having an arbitrary methylation rate can be obtained by adding 5-methyl dCTP to the reaction solution. The method for preparing methylated standard DNA by PCR is described below.

  In the PCR method, if any specific amount of 5-methyl dCTP is mixed in the PCR reaction solution, DNA in which the cytosine base portion of the PCR product DNA is converted to 5-methylcytosine can be obtained. In the PCR reaction, the position on the DNA strand into which 5-methylcytosine is incorporated cannot be specified, and cytosine or 5-methylcytosine enters randomly. That is, in the PCR reaction, cytosine at any position becomes 5-methylcytosine regardless of the -CG-continuous sequence on the template DNA. Of course, if only 5 methyl dCTP is used without adding dCTP during the PCR reaction, DNA in which all the cytosine in the DNA product obtained by PCR is converted to 5-methyl cytosine can be obtained. In the PCR reaction using Taq DNA polymerase, the uptake efficiency of dCTP and 5-methyl dCTP hardly changes. Therefore, when the PCR reaction of dCTP and 5-methyl dCTP is performed at a ratio of 1: 1, the rate of incorporation of cytosine and 5-methylcytosine in the DNA product obtained by the PCR is determined under optimal conditions. If done, it can be made about 1: 1 to 2: 1. Compared to other fluorescently labeled nucleotides, 5-methyl dCTP has a molecular structure similar to that of dCTP, so it has better uptake efficiency and can replace all cytosines in PCR products with 5-methyl cytosine. It is. In the PCR reaction in which all of cytosine in the PCR reaction is replaced with 5-methylcytosine, it is necessary to slightly change settings such as the annealing temperature and time in the PCR reaction. Actually, it is necessary to amplify by setting the annealing temperature as low as 2 ° C. to 5 ° C. However, the methylation rate of cytosine in the DNA to be analyzed in the present invention is usually 0% to 50%, and in the case of DNA obtained from an animal, the DNA methylation rate is usually 0% to 10%. For this reason, the methylated standard DNA used at this time is at most estimated to require DNA in which 50% cytosine is replaced with 5-methylcytosine. When dCTP and 5-methyl dCTP are added at a ratio of 1: 1 to 1: 2 in the PCR reaction solution so that the methylation rate of cytosine in the DNA of the PCR product is 50%, the temperature and time of the PCR reaction The PCR amplification product DNA can be obtained under substantially the same conditions as in the PCR reaction when 5-methyl dCTP is not mixed. However, in this case, the DNA yield is about 30% to 70%.

For example, an example of a method for preparing a methylated DNA standard for performing methylation analysis in the vicinity of the transcription start point of the human E-cadherin genomic DNA sequence will be described.
Template DNA: Human genomic DNA 1μg
Forward primer: 5'-ATAACCCACCTAGACCCTAGCAAC-3 '(SEQ ID NO: 8) 50pmole
Reverse primer: 5'-GAGTCTGAACTGACTTCCGCA-3 '(SEQ ID NO: 9) 50pmole
Taq DNA polymerase: TaKaRa Taq Hot Start Version (manufactured by Takara Bio Inc.), 2.5 unit nucleotides: 2.5 μmole each of dATP, dGTP, and dTTP
Cytosine: dCTP and 5-methyl dCTP added so that the total amount is 2.5 μmole
Bring the total volume of the PCR reaction system to 50 μL.
PCR reaction conditions:
(95 ° C, 2 minutes) x 1 cycle (95 ° C, 30 seconds / 59 ° C, 30 seconds / 72 ° C, 30 seconds) x 30 cycles (72 ° C, 8 minutes) x 1 cycle

を有する。前記配列は、ＰＣＲ産物である２本鎖ＤＮＡのうちのセンス鎖のみを記している。このセンス鎖のうち、四角で囲まれたオリゴＤＮＡプライマー部分を除く領域の塩基配列中のシトシンの位置に、任意に設定した割合の５−メチルｄＣＴＰがランダムの位置に取り込まれる。アンチセンス鎖も同様に、プライマー部分を除く領域に５−メチルｄＣＴＰがセンス鎖と同様の割合で取り込まれることとなる。
例えば、ＰＣＲ反応液中にｄＣＴＰと５−メチルｄＣＴＰとを９対１の割合で添加した場合には、上記塩基配列中の全シトシンのうちのおよそ１０％が５−メチルシトシンに転換される。このときの５−メチルシトシンの取り込まれる位置はランダムである。 The DNA amplified under the above conditions has the following base sequence (SEQ ID NO: 10):

Have The sequence describes only the sense strand of the double-stranded DNA that is a PCR product. Of this sense strand, an arbitrarily set proportion of 5-methyl dCTP is incorporated at random positions into the cytosine positions in the base sequence in the region excluding the oligo DNA primer portion surrounded by a square. Similarly, in the antisense strand, 5-methyl dCTP is incorporated into the region excluding the primer portion at the same rate as the sense strand.
For example, when dCTP and 5-methyl dCTP are added at a ratio of 9 to 1 in the PCR reaction solution, approximately 10% of the total cytosine in the base sequence is converted to 5-methylcytosine. At this time, the position where 5-methylcytosine is taken in is random.

  The methylated standard DNA prepared as described above is prepared and treated with bisulfite ion in the same manner as the genomic DNA to be analyzed for the methylation rate. In order to quantify the cytosine methylation of DNA to be analyzed, methylated standard DNAs having various methylation rates are prepared in advance. Next, both the methylated standard DNA and the methylation rate analysis target DNA were subjected to bisulfite ion treatment under the same conditions. Hereinafter, DNA amplification, preparation of single-stranded DNA, and labeling analysis were performed. Perform the operation.

(7) Double-stranded non-separation type measurement method In the measurement method of the present invention, the double-stranded DNA obtained in the amplification step is not separated into a sense strand and an antisense strand, and the double-stranded DNA remains in a double-stranded DNA state The amount of labeled cytosine or labeled guanine can be measured.

標的配列がメチル化されていない場合、亜硫酸水素イオン処理したＤＮＡをＰＣＲ増幅したＤＮＡ産物の内のセンス鎖にはシトシンは含まれないが、アンチセンス鎖にはセンス鎖のグアニン数と等しい数のシトシンが存在し、ＰＣＲで得られたＤＮＡ産物中のアンチセンス鎖のシトシン数は、亜硫酸水素イオン処理前の標的ＤＮＡのシトシンのメチル化率に係わらず、常に一定である。ＰＣＲで得られたＤＮＡ産物中のアンチセンス鎖のシトシンは標的配列のメチル化に係わらず常に５個で一定である（下記参照）。これに対してセンス鎖中に存在するシトシンは標的ＤＮＡのメチル化率に応じて変化する。 Hereinafter, description will be made assuming that the base sequence between the PCR primers is as follows (the primer portion is omitted). The cytosine in the boxed portion is cytosine that can be methylated. The following DNA is the target DNA sequence prior to bisulfite ion treatment.

When the target sequence is not methylated, the sense strand in the DNA product obtained by PCR amplification of DNA treated with bisulfite ion does not contain cytosine, but the antisense strand has a number equal to the number of guanines in the sense strand. In the presence of cytosine, the number of cytosines in the antisense strand in the DNA product obtained by PCR is always constant regardless of the cytosine methylation rate of the target DNA prior to bisulfite ion treatment. The antisense strand cytosine in the DNA product obtained by PCR is always constant at 5 regardless of the methylation of the target sequence (see below). In contrast, cytosine present in the sense strand varies depending on the methylation rate of the target DNA.

センス鎖で四角で囲んだＴは、標的ＤＮＡ配列のセンス鎖においてメチル化されていないシトシンが、Ｔに変換された箇所を示す。センス鎖のシトシンは０個であり、アンチセンス鎖のシトシンは５個である。従って、亜硫酸水素イオン処理及びＰＣＲ処理の後では、標的二本鎖ＤＮＡ中のシトシンの数は合計５個である。 (7-i) When 0 cytosine is methylated The base sequence of the double-stranded DNA product after bisulfite ion treatment and PCR treatment is as follows:

T surrounded by a square in the sense strand indicates a site where cytosine which is not methylated in the sense strand of the target DNA sequence is converted to T. There are 0 cytosines in the sense strand and 5 cytosines in the antisense strand. Therefore, after bisulfite ion treatment and PCR treatment, the total number of cytosines in the target double-stranded DNA is 5.

センス鎖で四角で囲んだＣは、標的ＤＮＡ配列のセンス鎖においてメチル化されていたシトシンの箇所を示す。センス鎖のシトシンは３個であり、アンチセンス鎖のシトシンは５個である。従って、亜硫酸水素イオン処理及びＰＣＲ処理の後では、標的二本鎖ＤＮＡ中のシトシンの数は合計８個である。 (7-ii) When cytosine at three sites is methylated The base sequence of the double-stranded DNA product after bisulfite ion treatment and PCR treatment is as follows:

C surrounded by a square in the sense strand indicates a site of cytosine that has been methylated in the sense strand of the target DNA sequence. There are 3 cytosines in the sense strand and 5 cytosines in the antisense strand. Therefore, after bisulfite ion treatment and PCR treatment, the total number of cytosines in the target double-stranded DNA is 8.

From the above relationship, when the number of methylated cytosines contained in the sense strand of the target DNA sequence in the sample DNA to be amplified by PCR is mc, it is expressed by the following formula:

a = (Sg−Pg) + (c + mc−Pc)

Pc: the number of cytosines contained in the forward primer.
Pg: the number of cytosines contained in the reverse primer.
c: Number of unmethylated cytosines contained in the sense strand of the target DNA sequence in the sample DNA to be amplified by PCR.
mc: the number of methylated cytosines contained in the sense strand of the target DNA sequence in the sample DNA to be amplified by PCR.
Sg: the number of guanines contained in the sense strand of the target DNA sequence in the sample DNA to be amplified by PCR.
a: The number of cytosines that can incorporate fluorescently labeled dCTP in the double-stranded DNA product obtained by PCR (total number of both strands of the sense strand and the antisense strand).

In the above formula, when a is ₀ when mc = 0,

a = mc + a ₀

Thus, the variable a increases by the number of methylated cytosines on the sense strand of the target DNA (in the case where G is included in the PCR primer, C is incorporated into the complementary DNA region). Therefore, the number of methylated cytosines on the sense strand of the target DNA can be calculated even if the total number of labeled cytosines contained in both the sense strand and the antisense strand is measured in the double-stranded DNA state. it can.
More specifically, the labeled double-stranded DNA or single-stranded DNA is degraded to the nucleotide level by nuclease P1, and the number of fluorescently labeled nucleotide molecules contained in the enzyme digest is degraded by the enzyme. The number count of fluorescence and the number count of single molecule fluorescence can be analyzed using, for example, Olympus MF20.

(8) Measurement method using SSCP (single strand conformational polymorphism) electrophoresis The measurement method of the present invention can be obtained by amplification because PCR amplification can be performed using one or two types of fluorescently labeled nucleotides. The double-stranded DNA is separated by SSCP electrophoresis, and the DNA product can be analyzed by a fluorescence scanner with the gel remaining in the glass plate after the electrophoresis. When SSCP electrophoresis is used, the time from DNA amplification to methylation analysis can be greatly shortened.

In addition, DNA products obtained by PCR using nucleotides labeled with different types of fluorescent substances of dCTP and dGTP, and using bisulfite ion-treated DNA as a template are biased to the sense strand and the antisense strand, respectively. The nucleotide is incorporated.
Specifically, in higher animals, cytosine in a continuous sequence of -CG- is often methylated, so that other cytosine is converted to uracil by bisulfite ion treatment, and subsequent PCR amplification The DNA treated with bisulfite ion serves as a template and is converted into thymine in the PCR product. Therefore, when cytosine remains in the sense strand of the PCR product, it means that the cytosine has been methylated. For example, when three cytosines remain in the sense strand in the PCR product, there are three guanines complementary to cytosine in the antisense strand.

  On the other hand, the guanine of the sense strand and the cytosine of the antisense strand are constant regardless of the degree of methylation of the template DNA. Therefore, the “ratio of moles of cytosine to moles of guanine in the sense strand” and “ratio of moles of guanine to moles of cytosine in the antisense strand” are equal, and the methylation rate of the DNA sample to be analyzed Can be calculated accurately by obtaining the ratio. In the method using SSCP in the present invention, the methylation rate is not predicted by the mobility of single-stranded DNA in the gel, but double-stranded DNA is separated in the gel (strand separation) and the respective bands. By obtaining the ratio of the two types of fluorescence for each, it was possible to accurately quantify.

  Further, even if two kinds of fluorescently labeled nucleotides are not used as described above, DNA amplification method (especially PCR amplification method) is performed by using one kind of fluorescently labeled nucleotide, for example, fluorescently labeled dCTP or fluorescently labeled dGTP. It is also possible to quantify the methylation rate by subjecting DNA, which is an amplification product obtained using a sample as a template, to an electrophoresis gel and comparing the fluorescence between bands of each single-stranded DNA of different samples. For example, when labeled dCTP is used, since the number of cytosines in the antisense strand is constant, the methylation rate can be quantified by measuring the amount of cytosine in the sense strand based on the amount of cytosine in the antisense strand. Is possible. Alternatively, when labeled dGTP is used, since the number of guanines in the sense strand is constant, the methylation rate can be quantified by measuring the amount of guanine in the antisense strand based on the amount of guanine in the sense strand It is.

(9) Measurement method using mass spectrometer As described above with respect to the analysis step, in the analysis step of the measurement method of the present invention, the molecular weight after binding the partner can be obtained without labeling a partner that can specifically bind to the labeling substance. For example, by measuring with a mass spectrometer, the binding amount of the partner to the labeling substance can be calculated, and the amount of labeled cytosine can be determined. However, the mass spectrometer currently has a measurement limit that can only read the molecular weight of low molecular weight DNA (for example, about 100 bp to 300 bp). Therefore, when the PCR product has a high molecular weight, the molecular weight can be measured by decomposing the high molecular weight PCR product with a restriction enzyme and applying it to a mass spectrometer. In this case, it is possible to easily analyze a molecular weight that is as close as possible to the analysis target sample, rather than the size of the digested fragment of DNA by the restriction enzyme.

  As a restriction enzyme that fragments a DNA product obtained by DNA amplification (for example, PCR method) after bisulfite ion treatment, digestion (fragmentation) of the DNA with a restriction enzyme by methylation of DNA as a starting material It is preferable to use a restriction enzyme that is not influenced by, for example, a restriction enzyme that does not contain cytosine in the recognition site. Examples of such restriction enzymes include PshBI (ATTAAT), SspI (AATTATT), or SwaI (ATTTAAAT) (above, Takara), AsnI (ATTAAT), DraI (TTTAAA), or Tru9I (TTAA) (above, Stratagene), or Tsp509I (AATT) or MseI (TTAA) (NEB).

If cytosine that is not commonly methylated on the base sequence between different sample DNAs or cytosine that is commonly methylated is known in advance, it is not limited to the restriction enzyme, and the base sequence of that region is A restriction enzyme can be selected as the target region. The analysis is simpler if the size of the DNA fragment after restriction enzyme digestion is the same or approximate between a plurality of analysis samples.
In addition, when the size of the DNA fragment obtained by the restriction enzyme treatment differs due to the difference in the methylation of the sample, the peak representing each mass obtained by the mass analyzer is changed to a known peak of the DNA fragment. By analyzing in detail based on the base sequence information, it is possible to obtain the methylation rate of the sample to be analyzed.

（四角で囲んだ領域はＰＣＲ用プライマーに由来する配列で、この部分はメチル化されていない）
をメチル化率分析対象ＤＮＡとする場合、この配列のうちの５’−ＣＧ−３配列に含まれるシトシン以外は亜硫酸水素イオン処理とそれに続くＤＮＡ増幅工程によりチミンに置換されるので、５’−ＣＧ−３配列に含まれるシトシン以外は分析対象試料間で共通にチミンとして現れる。 For example, where the following sequence of the promoter region of human E-cadherin (SEQ ID NO: 14):

(The region enclosed by a square is a sequence derived from a PCR primer, and this part is not methylated)
Is the DNA to be analyzed for methylation rate, except for cytosine contained in the 5′-CG-3 sequence in this sequence, it is replaced with thymine by bisulfite ion treatment and the subsequent DNA amplification step. Except for cytosine contained in the CG-3 sequence, it appears as thymine in common among the samples to be analyzed.

例えば、制限酵素Ｔｓｐ５０９Ｉの認識配列である−ＡＡＴＴ−に注目すると、前記配列における−ＡＡＴＴ−配列はすべてＤＮＡのメチル化に左右されない認識部位となっている。すなわち、高等動物では−ＣＧ−の連続配列のうちのシトシンがメチル化される可能性があるが、前記条件に当てはまる−ＣＧ−配列を起源とするＡＡＴＴ配列は存在しない。このような酵素を選択してＤＮＡの制限酵素による断片化を行えば、分析対象ＤＮＡのメチル化率の如何によらず、前記制限酵素によって得られる各ＤＮＡ断片の分子量は異なる試料ＤＮＡ間においても近似している。従って、前記ＤＮＡ断片をそれぞれ詳しく質量分析することができ、各断片のメチル化率を正確に求めることが可能となる。もちろん、複数種類の制限酵素を組み合わせて行うことも可能である。 When the DNA to be analyzed is not methylated at all, the base sequence of the sense strand of the DNA obtained by the bisulfite ion treatment and the subsequent DNA amplification process is as follows (SEQ ID NO: 15).

For example, when attention is paid to -AATT-, which is a recognition sequence of the restriction enzyme Tsp509I, all of the -AATT-sequences in the sequence are recognition sites independent of DNA methylation. That is, in higher animals, cytosine in a continuous sequence of -CG- may be methylated, but there is no AATT sequence originating from the -CG- sequence that meets the above conditions. If such an enzyme is selected and DNA is fragmented by a restriction enzyme, the molecular weight of each DNA fragment obtained by the restriction enzyme varies between different sample DNAs regardless of the methylation rate of the DNA to be analyzed. Approximate. Therefore, each of the DNA fragments can be subjected to mass spectrometry in detail, and the methylation rate of each fragment can be accurately obtained. Of course, it is also possible to combine a plurality of types of restriction enzymes.

  Therefore, the restriction enzyme used when the PCR product has a high molecular weight is a restriction enzyme that does not contain cytosine in the recognition sequence of the restriction enzyme, or a DNA amplification reaction (such as PCR method) following bisulfite ion treatment. The nucleotide sequence of the DNA product obtained by performing the procedure does not contain thymine (T) derived from cytosine in the continuous sequence of -CG- of the nucleotide sequence of the sample DNA (before bisulfite ion treatment). Restriction enzymes that recognize are preferred.

[2] Cell or tissue of the present invention DNA methylation is a common method of maintaining transcription in a repressed state, and is an epigenetic mechanism that plays an important role in mammalian gene regulation (human molecular genetics). 2nd edition, p185, Publisher: Medical Science International). About half of the human genes have CpG islands [Ioshikhes IP and Zhang MQ, Nature Genetics, vol.26 (1), pp61-63, 2000], and said CpG islands methylate cytosine in cells. It is known as a base sequence that protects against the damage.

  That is, cytosine in a CpG island with a dense -CG-sequence is less susceptible to methylation than cytosine at other sites. Therefore, since genes having CpG islands in the promoter region are usually less susceptible to cytosine methylation modification, the genes include many housekeeping genes and genes that are widely expressed. However, recent studies have reported that abnormal methylation of cytosine in CpG islands occurs in certain cancer cells and aging cells [Cancer Research, vol. 63, pp1114-1121, 2003 Melki JR et al., Blood, vol. 15, No. 10, pp3208-3213, 2000; Leopoldo Alves Ribeiro-Filho et al., Molecular Carcinogenesis, vol. 34, pp187-198, 2002; Maria F. Paz et al., Cancer Research (USA), vol. 63, pp1114-1121, 2003; Toyota M. et al., Cancer Research, vol. 59, No. 1, pp5438-5442, 1999]. A gene having a CpG island in the core promoter region near the transcription start point of the gene tends to suppress downstream gene expression due to abnormal methylation of cytosine in the region. Examples of those in which the binding of the transcription factor to the target DNA is inhibited by cytosine methylation in the recognition sequence include, for example, E2F, CREB, AP2, cMyc, Myn, NF-κB, cMyb, ETS, Sp1, etc. Although there are other transcription factors, if the transcription factor contains cytosine in the target binding base sequence, some change occurs in the binding affinity with the transcription factor due to methylation of cytosine in the recognition sequence. Is easily guessed. That is, abnormal methylation of cytosine in the promoter region of a gene affects the balance of homeostasis. Abnormal hypermethylation of CpG islands is frequently observed when cells become cancerous [Maria F. Paz et al., Cancer Research (USA), vol. 63, pp1114-1121, 2003]. Since there are over 10,000 genes in humans, it is thought that cell homeostasis is destroyed by abnormal methylation as well as gene defects in cancer cells [Jones PA and Laird PW , Nature Genetics, vol.21 (2), pp163-167, 1999]. Even if there is no CpG island in the core promoter, if an abnormality occurs in cytosine methylation in a gene expression regulatory region such as an enhancer or silencer, the region is a region to which a transcription factor binds. It is easily inferred that disruption of homeostasis occurs as in the case of genes having islands. It has also been clarified that hypermethylation of cytosine induces chromatin structural changes, that is, deacetylation of histone, which is a component of chromatin, and suppresses transcription of the gene bound to the chromatin. . That is, the expression of a gene in which cytosine is highly methylated is suppressed by the above-mentioned two transcriptional control mechanisms, namely, the mechanism that prevents the binding of transcription factors to the hypermethylation of cytosine contained in the gene.

  By using the DNA methylation analysis method of the present invention, the methylation rate of a specific region involved in the regulation of gene expression related to the maintenance of cell homeostasis is compared with the methylation rate of a normal cell gene. Predicting the safety of the cells when the cells are treated by transplanting them into the living body, such as whether the target cells are normal cells or abnormal cells such as entering the canceration process Is possible. Thus, when transplanting useful cells into a living body such as a human, it is extremely important to compare the methylation rate of the genomic DNA of the cells with that of normal cells before transplanting the cells into the living body. is there. In the case of humans, the total number of genes is said to be approximately 32,000. However, it is not necessary to analyze the methylation of all the genes, and it is not more than several thousand, preferably not more than 1,000, more preferably It is sufficient to analyze the cytosine methylation of 500 or less, more preferably 100 or less, more preferably 10 or less, more preferably several or less genes. This is based on the grounds that cytopathic methylation abnormalities of only a few specific genes are not observed due to canceration or aging, but the methylation abnormalities are observed in a wide range of genes [Jones PA and Laird PW, Nature Genetics, vol. 21 (2), pp163-167, 1999; Maria F. Paz et al., Cancer Research (USA), vol.63, pp1114-1121, 2003]. Therefore, it is not necessary to perform cytosine methylation analysis for all genes in order to evaluate the safety of cells to be examined for genomic DNA. Methylation can be inferred and it can be statistically inferred whether the cell is a normal cell.

  For example, taking a tumor suppressor gene as an example, by comparing the methylation rate of a CpG island of a gene having a CpG island in the promoter region of the gene with that of a normal cell, whether or not the cell is normal is determined. Can be determined.

  For example, regarding cell safety evaluation criteria when embryonic stem cells (ES cells) and tissue stem cells are cultured, artificially differentiated into neurons in vitro, and further transplanted in vivo explain. When stem cells are artificially differentiated into target nerve cells and further transplanted in vivo, the cells are several thousand or less, preferably 1,000 or less, more preferably 500 or less, and still more preferably 100. Less than, more preferably 10 or less, more preferably 1 cytoplasmic methylation of one gene is analyzed, and the DNA methylation rate of the gene group is compared with the DNA methylation of a normal neuronal gene group. The cells of the present invention can be obtained by comparing with the differentiated nerve cells.

  In normal cells, there is usually a region where the cytosine methylation rate is low, and conversely, there is usually a region where cytosine is highly methylated, which also varies depending on the cell type. A typical example of the former is a CpG island, but generally, the methylation rate of the region is said to be low. Representative examples of the latter include parasite genes derived from microorganisms integrated into genomic DNA, retrotransposons, etc., and the expression of the genes is harmful to the host, and is usually highly methylated in the genome. Has been inactivated. In order to determine whether or not the cell to be examined is normal, it is only necessary to analyze whether or not the methylation of the genomic DNA of the cell approximates that of a normal cell, and the methylation rate of the DNA is in the normal range. If it is within the range, it is determined that the cell to be examined is normal, and the cell is safe, so that the cell can be safely transplanted into a living body. The criteria for determining the safety of the following cells to be examined from methylation of genomic DNA and the basis for the setting are described below.

  Methylation of analysis target genes of cells to be tested The methylation rate of cytosine present in the analysis target region, particularly cytosine in the CpG island is 50% or more, and more than half of the genes to be tested are When the methylation rate is exceeded, the cells to be analyzed are similar to the characteristics of cancer cells, and it can be determined that the homeostasis of the cells is not maintained. It can be judged that it is a dangerous cell not suitable for transplantation [Maria F. Paz et al., Cancer Research (USA), vol. 63, pp1114-1121, 2003]. Therefore, cells other than those determined to be dangerous because the homeostasis is not maintained by the methylation rate test can be used as candidates for cells that can be transplanted into a living body.

  On the other hand, genes that are normally inactivated by cytosine methylation in genomic DNA include parasite genes derived from microorganisms integrated into genomic DNA, retrotransposons, and the like. In addition, promoter regions such as cell proliferation-related genes in cells of the nervous system are also highly methylated, and cell proliferation is suppressed. In this way, the genes whose genes are highly methylated and inactivated in normal cells are controlled so that they do not work in normal cells. Is considered to be safe as a cell to be transplanted into a living body. Specifically, in a normal cell, a cell in which the methylation rate of a DNA region to be analyzed for a specific methylation is usually 50% or less of the methylation rate of a normal cell is determined not to maintain homeostasis. It can be determined that the cells are dangerous cells that are not suitable for transplantation into a living body.

  In the production of cloned animals, DNA methylation abnormalities have been reported even in abnormal fetal growth resulting from epigenetic abnormalities. Therefore, when creating a cloned animal, the blood of the mother or fetus is collected and the methylation of a specific gene is compared with the normal case to determine whether the fetus is normal before birth. It is also possible to judge.

The methylation analysis target region of the analysis target gene of the analysis target cell or tissue is set to 50 to 2000 bp, more preferably 40 to 100 bp as an methylation analysis target region of any region of the gene. This is preferable. In particular, it is preferable that the analysis target region is a region between 5 bp (-5 kbp) upstream of the transcription start point of the gene and 100 bp (+100 bp) downstream of the transcription start point.
By analyzing the region of the gene by the methylation analysis method of the present invention, it can be determined whether the gene functions normally in the cell or is normally inactivated.

More specifically, in the case of genes that are transcribed in normal cells, the following are considered as dangerous areas. That is, when the cytosine methylation rate of the test target DNA of normal cells corresponding to the type of test target cell is 0% to less than 50%, the risk range of the methylation rate is 80% or more. In addition, when the methylation rate of cytosine in the test target DNA of normal cells corresponding to the type of test target cell is 50% or more, the risk range of the methylation rate is 90% or more.
Furthermore, genes that are normally not transcribed in normal cells, such as parasite genes derived from microorganisms or retrotransposons, or genes that are toxic or harmful to cells or organisms when transcribed, are usually highly methylated. In the case where the region to be subjected to methylation analysis is the target, the case where the methylation rate of cytosine in the region to be examined is 20% or less is regarded as a dangerous region.

  The cell of the present invention can be analyzed by extending its safety to the total number of genes of the cell to be examined. Preferably, the cell safety is analyzed by analyzing methylation of 2 to 1000 kinds of genes. Sex can be evaluated. In particular, in this case, it is determined that the cell to be examined is dangerous when it is determined that it is dangerous for 50% or more of the genes in a plurality of types of genes to be analyzed for methylation according to the risk criteria described above. Other cells are judged to be highly safe and can be made candidates for cells that can be transplanted into a living body.

[3] Evaluation method of the present invention According to the evaluation method of the present invention, the state of a cell can be determined. Examples of the cell state that can be determined by the evaluation method of the present invention include malignancy of tumor cells, differentiation state of cells, safety of cells for biological transplantation, aging of cells, etc. Can be distinguished from abnormal cells.

  In the evaluation method of the present invention, the use of an anti-methylated cytosine antibody as an antibody, the contact between a cell chromosome specimen and an anti-methylated cytosine antibody, dissociating a monovalent bond in an antigen-antibody reaction, and a bivalent bond Can be carried out in accordance with a normal immunostaining method, except that it is carried out under conditions that can maintain the above (hereinafter referred to as divalent binding conditions).

In antigen-antibody reactions, it is known that bivalent binding of antibodies exhibits an affinity about 10 ³ (M ⁻¹ ) times stronger than monovalent binding [eg, Ivan Roitt, Jonathan Brostoff, and David Male, translated by Tomio Tada, Immunology Illustrated (5th edition of the original), published February 10, 2000, Nanedo, page 110 (original name: IMMUNOLOGY, FIFTH EDITION)]. Here, the bivalent binding means that a single molecule antibody or a bimolecular antibody immobilized on one carrier binds to an antigen at two antigen binding sites. Means that one molecule of an antibody binds to an antigen at one antigen binding site.

This point will be further described with reference to FIGS. FIGS. 7 to 10 are schematic diagrams showing the state of binding between one molecule of double-stranded DNA (1) having protruding ends at both ends and one molecule of anti-methylated cytosine antibody (2). The black circle (FIGS. 7 to 10) described at the protruding end of the double-stranded DNA (1) means methylated cytosine, and the white circle (FIG. 7) means cytosine.
As shown in FIGS. 7 to 10, two antigen-binding sites exist in the single molecule antibody (2). In monovalent binding, one of the antigen-binding sites binds to double-stranded DNA (1), which is an antigen (FIG. 7). On the other hand, in bivalent binding, one molecule of antibody (2) binds to one molecule of antigen (1) using the two antigen binding sites (FIG. 8) or carrier (3). The two molecules of antibody (2) immobilized on each bind to one molecule of antigen (1) using one antigen binding site (FIG. 9). In FIG. 10, two molecules of antibody (2) bind to one molecule of antigen (1) using one antigen binding site, but the affinity in this case is the same as monovalent binding. It is known that

  As described above, since there is a difference in affinity between a bivalent bond and a monovalent bond, by changing various conditions in the antigen-antibody reaction system, the monovalent bond is dissociated and the bivalent bond is maintained. It is possible. The binding between antigen and antibody is based on, for example, binding by electrostatic force, hydrogen bonding, or hydrophobic bonding, and binding by electrostatic force is performed by, for example, salt concentration or pH, and hydrogen bonding is performed by, for example, urea or guanidine. It is known that the hydrophobic bond is affected by the concentration of hydrochloric acid, for example, by the concentration of polyethylene glycol.

For example, regarding a salt (eg, NaCl) concentration in an antigen-antibody reaction system, a bivalent bond is maintained, but a monovalent bond is excluded (note that a bivalent bond and a monovalent bond are mixed, and the ratio is 1 The conditions may vary depending on the type of antibody or salt used. For example, according to the method described in Reference Example 1, for each antibody used, the conditions may be changed. The salt concentration range can be easily determined.
In addition, the divalent bond is maintained in the same manner with respect to conditions other than the salt concentration that can affect the affinity, such as urea, guanidine hydrochloride, or polyethylene glycol concentration, or pH. The conditions under which binding is eliminated can be readily determined for each antibody used.

  When the contact between the cell chromosomal specimen and the anti-methylated cytosine antibody is carried out under divalent binding conditions, for example, the cell chromosomal specimen and the anti-methylated cytosine antibody are first subjected to the divalent binding conditions. It can also be carried out by contact, or after carrying out contact between a cell chromosome sample and an anti-methylated cytosine antibody under normal antigen-antibody reaction conditions, the chromosome sample under divalent binding conditions It can also be carried out by washing.

  The chromosome sample of the cell used in the evaluation method of the present invention is not particularly limited as long as the anti-methylated cytosine antibody can specifically react with methylated cytosine contained in the DNA constituting the chromosome, For example, it can be prepared according to a normal method for preparing a chromosome sample, and for example, a cell chromosome sample can be prepared according to the procedure described in Example 3.

  In the evaluation method of the present invention, a cell specimen used in a normal immunostaining method can be used as it is, but it is preferable to carry out the next antibody reaction after protease treatment of the cell specimen. It is more preferable to perform the next antibody reaction after further acid treatment and / or alkali treatment after the treatment.

  Examples of the protease used for the protease treatment include trypsin and pepsin. The protease (eg, trypsin) concentration can be appropriately determined according to, for example, the treatment temperature or the treatment time, and is, for example, 0.01 to 0.5%, preferably 0.02 to 0.1%. Can be implemented. The treatment temperature can be, for example, 0 ° C. (for example, on ice) to room temperature, preferably 0 to 10 ° C. The treatment time can be, for example, 5 seconds to 30 minutes, preferably 15 seconds to 10 minutes, more preferably 30 seconds to 5 minutes. Although the present invention is not limited to the following inference, by carrying out protease treatment, the protein on the chromosome is removed, and the reaction between the anti-methylated cytosine antibody and DNA in the next antibody reaction is promoted. It is considered a thing.

  The acid treatment can be performed using, for example, an inorganic acid (for example, hydrochloric acid, sulfuric acid, or nitric acid) or an organic acid. The concentration can be appropriately determined according to, for example, the processing temperature (usually room temperature) or the processing time, and is, for example, 0.1 to 5 mol / L, preferably 0.5 to 3 mol / L. be able to. The treatment time can be, for example, 1 minute to 1 hour, preferably 5 to 15 minutes. Although the present invention is not limited to the following inference, by performing acid treatment, depurination of DNA (deadenine or deguanine) occurs, and the anti-methylated cytosine antibody and DNA in the next antibody reaction It is considered that the reaction is promoted.

The alkali treatment can be performed using, for example, an aqueous solution of an alkali metal (for example, sodium or potassium) hydroxide. In the case of using an alkali metal hydroxide, the concentration can be appropriately determined according to, for example, the treatment temperature (usually room temperature) or the treatment time, but for example, 10 mmol / L to 2 mol / L, Preferably it can implement at 10-100 mmol / L. The treatment time can be, for example, 1 minute to 1 hour, preferably 5 to 15 minutes.
Only one of the acid treatment and the alkali treatment can be carried out, or can be carried out sequentially in any order.

Although the evaluation method of this invention is not limited to the procedure shown below, For example, it can implement by the following procedures.
Chromosome specimens of cells are blocked with an appropriate blocking agent (eg, 1% bovine serum albumin) (eg, 30 minutes at room temperature). After washing the chromosome sample and removing the blocking agent, the anti-methylated cytosine antibody is reacted as a primary antibody under normal antigen-antibody reaction conditions (for example, in the presence of 150 mmol / L NaCl) (for example, at room temperature). 1 hour). After removing the unreacted primary antibody by washing the chromosome sample, the chromosome sample is further washed under divalent binding conditions (for example, in the presence of 500 mmol / L NaCl) and bound to the chromosome sample by monovalent binding. Remove the primary antibody. Subsequently, the labeled (for example, fluorescently labeled) secondary antibody is reacted (for example, at room temperature for 30 minutes), and then the unreacted labeled secondary antibody is removed by washing. The chromosome specimen is subjected to detection means (for example, a fluorescence microscope) according to the label, and the stained image is observed.

  As a labeling substance for the secondary antibody, a substance that can be usually used for immunostaining, for example, a fluorescent substance, a luminescent substance, or an isotope can be used. Further, instead of using a labeled secondary antibody, the primary antibody can be directly labeled. Alternatively, instead of directly labeling the secondary antibody, the secondary antibody is modified with one of the specifically bindable partners (eg, biotin in biotin-avidin) and the other partner (eg, avidin) is labeled. You can also

In the evaluation method of the present invention, the state of the cell is determined from the obtained stained image. In immunostaining in which an antigen-antibody reaction is performed under normal physiological conditions (for example, NaCl concentration = 150 mmol / L), chromosomes are stained throughout in most cells including normal cells and cancer cells. There is almost no difference from the cells (for example, FIGS. 12 and 13 in Example 3).
On the other hand, in immunostaining in which an antigen-antibody reaction is performed under a bivalent binding condition, a staining positive portion appears as a minute spot (spot) on the chromosome. In cancer cells, the small spots are small (for example, FIGS. 14 and 15 in Example 3), and in normal cells, the spots are large (for example, FIGS. 16 and 17 in Example 3). . In addition, the number of spots is large in cancer cells (for example, FIGS. 14 and 15 in Example 3) and small in normal cells (for example, FIGS. 16 and 17 in Example 3). Therefore, by comparing the size and number of spots on the chromosome, it is possible to easily determine whether the test cell is a normal cell or a cancer cell.
The size of the spot varies depending on, for example, the type of marker used or the processing conditions for visualization thereof (for example, exposure time), and therefore the spot size can always be defined by an absolute value. is not. Therefore, preferably, a cell whose cell state is known in advance (more preferably, both normal cells and cancer cells are used) is used as a standard cell, and the size of the spot observed in the cell to be evaluated is By comparing with the size of the spot observed in the standard cell, the size of the spot of the evaluation target cell can be relatively determined, and the cell state can be evaluated based on the result. As the standard cells, it is preferable to use cells that are the same as or similar to the cells to be evaluated. For example, when evaluating the cell state of a cell collected from a certain tissue (for example, liver) or its established or differentiated cell, a cell collected from the same tissue or its established or differentiated cell, or derived from the tissue Cancer cells and the like can be combined as appropriate and used as standard cells.

EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
Example 1: Analysis of methylation of human cadherin promoter region
(1) Preparation of methylated DNA standard sample In this example, double-strand separation methylation analysis by SSCP was carried out using FITC-labeled dCTP and Texas Red-labeled dGTP, targeting the human cadherin promoter region. did.
Prior to the methylation analysis, three types of methylated DNA standards having cytosine methylation rates of 0%, 10%, and 25% were prepared as samples for methylation analysis according to the following procedure.

The PCR reaction for obtaining three types of methylated DNA standards uses human genomic DNA (1 μg) as template DNA, and each base represented by SEQ ID NO: 8 and SEQ ID NO: 9 as a forward primer and a reverse primer, respectively. Each oligonucleotide consisting of a sequence (50 pmole each) was used, 2.5 units of commercially available polymerase (TaKaRa Taq Hot Start Version; manufactured by Takara Bio Inc.) was used as Taq DNA polymerase, and dATP, dGTP, and dTTP were used as nucleotides. (2.5 μmole each), and dCTP and 5-methyl dCTP (2.5 μmole as the total amount) were used, and the total amount of the PCR reaction system was 50 μL. PCR reaction conditions were 95 ° C for 2 minutes, 30 cycles of 95 ° C for 30 seconds / 59 ° C for 30 seconds / 72 ° C for 30 seconds, and finally at 72 ° C. The reaction was performed for 8 minutes. The amount ratio (dCTP: 5-methyl dCTP) of dCTP and 5-methyl dCTP (total amount: 2.5 μmole) is 3: 1 when the methylation rate is 25%, and when the methylation rate is 10%. When the methylation rate was 9%, only dCTP was used.
By the PCR reaction, three types of methylated DNA standard products having the base sequence represented by SEQ ID NO: 10 were obtained.

(2) Methylation analysis of methylated DNA standard products The three types of methylated DNA standard products obtained in Example 1 (1) were subjected to bisulfite ion treatment according to the following procedure.
2 μg of each methylated DNA standard product was dissolved in 50 μL of sterilized distilled water, 5.5 μL of 3 mol / L sodium hydroxide solution was added, and the mixture was incubated at 42 ° C. for 20 minutes. Further, 500 μL of sodium bisulfite solution was added to the DNA solution and reacted at 55 ° C. for 6 hours. The sodium hydrogen sulfite solution (prepared at the time of use) was prepared by adding 8 mL of saturated sodium hydrogen sulfite solution adjusted to pH 5.0 using 10 mol / L sodium hydroxide solution and 0.22 g of hydroquinone in 10 mL of distilled water. Was prepared by mixing 0.5 mL of a solution prepared by dissolving in distilled water.

  Thereafter, the converted DNA in the reaction solution was purified using a commercially available reagent (Wizard Plus Minipreps, DNA Purification System; Promega, USA) according to the product manual. 5.5 μL of a 3 mol / L sodium hydroxide solution was added to 50 μL of the DNA solution obtained by the purification operation, and kept at 37 ° C. After reacting for 30 minutes, 66 μL of a 5 mol / L ammonium acetate solution and 0.5 μL of etatinate (manufactured by Nippon Gene) were added and mixed, and then 300 μL of 95% ethanol was further added and cooled at −30 ° C. for 20 minutes. The solution was centrifuged at 10,000 × g for 20 minutes at 4 ° C., then the supernatant was discarded, the precipitate containing DNA was rinsed lightly with 80% ethanol precooled to −30 ° C., and the precipitate was dried under reduced pressure. . The obtained DNA was dissolved in 30 μL of 10 mmol / L Tris / 1 mmol / L-EDTA (pH 8.0).

  Subsequently, 20 ng of methylated standard DNA treated with bisulfite ion was used as a template, and each oligonucleotide consisting of each base sequence represented by SEQ ID NO: 6 and SEQ ID NO: 7 (50 pmole each) as a forward primer and a reverse primer, respectively. And 2.5 units of commercially available polymerase (TaKaRa Taq Hot Start Version; manufactured by Takara Bio Inc.) as Taq DNA polymerase, dATP and dTTP (each 2.5 μmole), dCTP and FITC-labeled dCTP as nucleotides (Total amount 2.5 μmole) and dGTP and Texas Red labeled dGTP (total amount 2.5 μmole) were used, and the PCR reaction system total amount was 50 μL. PCR reaction conditions were 95 ° C for 2 minutes, 30 cycles of 95 ° C for 30 seconds / 59 ° C for 30 seconds / 72 ° C for 30 seconds, and finally at 72 ° C. The reaction was performed for 8 minutes. The ratio of dCTP and FITC-labeled dCTP (total amount 2.5 μmole) (dCTP: FITC-labeled dCTP) was 10: 1, and the ratio of dGTP and Texas Red-labeled dGTP (total amount 2.5 μmole) (dGTP: Texas Red labeled dGTP) was 10: 1.

  The obtained three types of PCR products (each 20 μL) were mixed with an equal amount of dimethylformamide, treated at 98 ° C. for 5 minutes, and immediately iced, followed by 8% acrylamide [acrylamide: N, N′-methylene. Electrophoresis was performed on a gel containing bisacrylamide = 49: 1 (weight ratio)] and 1 × TBE [Tris-base (109 g), boric acid (57 g), and EDTA-2Na (9.3 g)].

  FIG. 4 shows the results of electrophoresis using the methylated DNA standard product having a methylation rate of 25%. In FIG. 4, the left lane is the result of detecting a band based on FITC-derived fluorescence (green), and the center lane is the result of detecting a band based on Texas red-derived fluorescence (red). The right lane shows the result of detecting a band based on the fluorescence derived from FITC and Texas Red.

  In FIG. 4, the region of band A surrounded by a square contains sense strand DNA incorporating both FITC-labeled dCTP and Texas Red-labeled dGTP. That is, the fluorescence amount of FITC contained in the band A reflects the cytosine content contained in the sense strand. In addition, the fluorescence amount of Texas Red contained in band A reflects the guanine content in the sense strand. Since the amount of guanine fluorescence in the sense strand of the fluorescence-labeled DNA, which is the PCR product obtained in the analysis operation, does not change even if the DNA methylation analysis sample changes, the fluorescence amount of Texas Red is used as the internal standard. As described above, it is possible to easily calculate the cytosine content in the band A by measuring the amount of FITC fluorescence in the sense strand.

  On the other hand, the fluorescence amount of Texas Red in the antisense strand of band B, that is, the guanine content directly reflects the cytosine content of the sense strand. Similarly, the fluorescence amount of FITC in band B, that is, the cytosine content directly reflects the guanine content of the sense strand. Therefore, in the antisense strand of band B, in the same manner as in the sense strand of band A, the fluorescence amount of Texas Red of the antisense strand was measured using the fluorescence amount of cytosine FITC as an internal standard, It is also possible to easily calculate the guanine content of band B in the same manner as in the sense strand, thereby obtaining the cytosine content of the sense strand.

The method described above is a method for determining the cytosine content of the sense strand by measuring two types of different fluorescence contained in band A or band B, but one type of fluorescence contained in band A and band B is obtained. By measuring, the cytosine content of the sense strand can be calculated in the same manner as described above. The details are described below.
In FIG. 4, there is a large difference in the amount of FITC existing in the band A and band B regions. This is because approximately 25% of the total number of cytosines contained in the DNA to be measured in the measurement is substituted with methylated 5-methylcytosine. As a result, in band B and band A, There is a large difference in the amount of fluorescence of FITC. On the other hand, since the number of cytosines in the antisense strand corresponds to the number of guanines in the amplified base sequence, this is not the case even when samples with different cytosine methylation rates are used. Even so, as long as the base sequences are the same, the number of cytosines in the antisense strand is always constant. Based on this principle, in the above methylation analysis experiment, if the fluorescence amount of the FITC of the sense strand is calculated using the fluorescence amount of the FITC of the antisense strand as an internal standard, each electrophoresis Even if there is a variation in the amount of DNA after PCR amplification between samples, it is possible to calculate the methylation rate of cytosine in the measurement target nucleotide sequence based on the internal standard as described above.
In addition, because of the principle described above, if the amount of guanine fluorescence in the sense strand of the base sequence after amplification is used as an internal standard and the amount of guanine fluorescence in the sense strand is measured, PCR amplification between each sample in electrophoresis Even if there is a variation in the amount of DNA later, the methylation rate of cytosine in the measurement target nucleotide sequence can be calculated based on the internal standard as described above.

  The results of measuring the fluorescence intensity of each band shown in FIG. 4 and the fluorescence intensity of each band obtained by electrophoresis when using methylated DNA standards with methylation rates of 0% and 10% were measured. The results are shown below. In addition, the measurement was performed using FM-BIO (manufactured by Takara Bio Inc.). The fluorescence intensity in each table shown below is an example of an actual measurement value of the fluorescence intensity of each band obtained by the measuring device.

Table 3 shows the measurement results in the case of “0% methylated standard DNA” in which cytosine is not methylated at all.
<< Table 3 >>
Fluorescence intensity Fluorescence intensity
FITC (Cytosine) Texas Red (Guanine)
The sense strand 1.93x10 ⁶ 52.7 x10 ⁶
Antisense strand 42.2 x10 ⁶ 2.05x10 ⁶

From the data in Table 3, the ratio of the sense strand fluorescence amount to the antisense strand fluorescence amount in FITC is 1.93 / 42.2 = 0.0457 (1).
The ratio of the antisense strand fluorescence amount to the sense strand fluorescence amount in Texas Red is 2.05 / 52.7 = 0.0389 (2)
Met.

Table 4 shows the measurement results in the case of “10% methylated standard DNA” in which 10% of cytosine is methylated.
<< Table 4 >>
Fluorescence intensity Fluorescence intensity
FITC (Cytosine) Texas Red (Guanine)
The sense strand 5.21x10 ⁶ 48.2 x10 ⁶
Antisense strand 41.2 x10 ⁶ 6.42x10 ⁶

From the data of Table 4, the ratio of the fluorescence amount of the sense strand to the fluorescence amount of the antisense strand in FITC is 5.21 / 41.2 = 0.126 (3)
The ratio of the antisense strand fluorescence amount to the sense strand fluorescence amount in Texas Red is 6.42 / 48.2 = 0.133 --- (4)
Met.

Table 5 shows the measurement results in the case of “25% methylated standard DNA” in which 25% of cytosine is methylated.
<< Table 5 >>
Fluorescence intensity Fluorescence intensity
FITC (Cytosine) Texas Red (Guanine)
Sense strand 10.8 x10 ⁶ 50.7 x10 ⁶
Antisense strand 40.8 x10 ⁶ 14.2 x10 ⁶

From the data in Table 5, the ratio of the fluorescence amount of the sense strand to the fluorescence amount of the antisense strand in FITC is 10.8 / 40.8 = 0.265 (5)
The ratio of the fluorescence intensity of the antisense strand to the fluorescence intensity of the sense strand in Texas Red is 14.2 / 50.7 = 0.280 (6)
Met.

  In the measurement result, paying attention to only FITC, draw a calibration curve (Fig. 5) using the values calculated in (1), (3) and (5) above, and the methylation rate is unknown The methylation rate of the target DNA can be determined. Similarly, draw a calibration curve (FIG. 6) using the values calculated in (2), (4), and (6), focusing only on Texas Red in the measurement results, and methylate The methylation rate of DNA to be measured whose rate is unknown can also be determined. In the calculations of (1) to (6), the amount of fluorescence that changes depending on the methylation rate of the DNA to be measured is used as the numerator of the formula, and the amount of fluorescence that is a reference that does not depend on the methylation rate of the DNA to be measured is used. Calculate in the denominator of the formula.

  As shown in Tables 3 to 5 above, with the cytosine methylation rate, the ratio of the FITC fluorescence intensity of the sense strand to the FITC fluorescence intensity of the antisense strand, and the ratio of the antisense strand to the Texas Red fluorescence intensity of the sense strand The ratio of Texas Red fluorescence intensity increased, and this increase rate was proportional to the DNA methylation rate.

The substantial amount of amplified DNA subjected to electrophoresis is indicated by the amount of cytosine in the antisense strand, ie, in this case, the amount of FITC fluorescence in the antisense strand, and similarly, in the case of Texas Red This is indicated by the amount of guanine in the strand, that is, the fluorescence amount of Texas Red in the sense strand in this case.
This principle is due to the fact that the amount of cytosine in the antisense strand of the amplified DNA does not depend on the methylation rate of the DNA to be measured, and similarly, the amount of guanine in the sense strand does not depend on the methylation rate of the DNA to be measured. It is.
Because of the principle as described above, even if there is a variation in the amount of each sample subjected to electrophoresis, pay attention to nucleotides that do not depend on the methylation rate of the DNA to be measured and use it as an internal standard. It is possible to correct the amount of fluorescence of nucleotides that varies with the methylation rate.

  In this measurement method, since the fluorescently labeled nucleotides are mixed at an arbitrary constant ratio in the DNA amplification step, the ratio of the fluorescence intensity of the two fluorescently labeled nucleotides in the sense strand or the antisense strand is used. The methylation rate of the DNA to be measured can be determined in the same manner as described above.

  Specifically, the ratio of the two fluorescence intensities in the sense strand or antisense strand is calculated as follows, and the methylation rate of the DNA to be measured is calculated. In this case, the actual value of the fluorescence amount of the fluorescently labeled nucleotide that changes depending on the methylation rate of the DNA to be measured is used as the denominator of the equation, and the fluorescence amount of the fluorescently labeled nucleotide that does not depend on the methylation rate of the DNA to be measured By placing the actual measurement value in the denominator of the equation, a value (right side of the equation) depending on the methylation of the DNA to be measured can be calculated.

In the case of 0% methylated standard DNA,
Sense strand 1.93 / 52.7 = 0.0366 (7)
Antisense strand 2.05 / 42.2 = 0.0486 --- (8)
It is.
In the case of 10% methylated standard DNA,
Sense strand 5.21 / 48.2 = 0.108 --- (9)
Antisense strand 6.42 / 41.2 = 0.156 --- (10)
It is.
In the case of 25% methylated standard DNA,
Sense strand 10.8 / 50.7 = 0.213 (11)
Antisense strand 14.2 / 40.8 = 0.348 --- (12)
It is.

  As shown in the above equation, a calibration curve can be written using the values of (7), (9), and (11), and (8), (10), and A calibration curve is drawn using the value of (12), and the methylation rate of the DNA to be measured, which is an unknown methylation rate, can be obtained. That is, it is possible to obtain the methylation rate of the DNA to be measured by simultaneously paying attention to the two fluorescence amounts in either the sense strand or the antisense strand in the electrophoresis.

  However, if the substantial DNA amount of the amplified DNA sample to be subjected to electrophoresis is accurately quantified in advance, it is not necessary to separate each DNA strand of the double-stranded DNA. In this case, the FITC emitted from the double-stranded DNA By determining the ratio between the fluorescence amount and the fluorescence amount of Texas Red, the methylation rate of the DNA to be measured can be quantified. In this case, the double-stranded DNA does not need to be electrophoresed, and the methylation rate of any DNA to be measured can be determined from the calibration curve by measuring the two types of fluorescence of the DNA. This principle has been described earlier in (7) Double-strand non-separated methylation measurement method.

<< Example 2: Methylation analysis of human integrin α2 promoter region by ELISA >>
Human blood was collected, and genomic DNA was purified from human leukocytes using a genomic DNA purification column (Qiagen) and fragmented with the restriction enzyme EcoRI according to a conventional method. Next, the DNA fragment was treated with bisulfite ion using a commercially available kit (CpGenome Modification Kit, Cat # S7820; manufactured by Intergen Company) to chemically convert unmethylated cytosine to uracil. The base-substituted DNA obtained by the conversion treatment was used as a template DNA, and an upstream primer for PCR with biotinylation at the 5 ′ end and a downstream primer for PCR were used, and further 20% of cytosine was 80%. PCR amplification was performed using a nucleotide mixture containing 5-methylcytosine (including 5-methyl dCTP, dCTP, dATP, dGTP, and dTTP). This replaces about 20% of cytosine in the PCR product DNA with methylated cytosine, ie, 5-methylcytosine, at random positions.

ＰＣＲ用フォワードプライマー（配列番号２５）：
５’− TTATTTAGGAAAAATAGAGAAAGGGA −３’ （５’末端はビオチン化してある）
ＰＣＲ用リバースプライマー（配列番号２６）：
５’− CCTAACAAACTTTCCTACCCTAAAC −３’
鋳型ＤＮＡ：ヒトゲノムＤＮＡ １μｇ
ヌクレオチド：ｄＡＴＰ、ｄＧＴＰ、及びｄＴＴＰをそれぞれ２．５μｍｏｌｅとし、更に、２．５μｍｏｌｅのｄＣＴＰと０．５μｍｏｌｅの５−メチルｄＣＴＰ（ｄＣＴＰと５−メチルｄＣＴＰの添加量合計が２．５μｍｏｌｅ）を使用。
ＰＣＲ反応系総量：５０μＬ
ＰＣＲ反応条件：
（９５℃，２分）ｘ１サイクル
（９５℃，３０秒／５９℃，３０秒／７２℃，３０秒）ｘ３０サイクル
（７２℃，８分）ｘ１サイクル Specific PCR conditions are shown below.
PCR target DNA sequence (SEQ ID NO: 24):

PCR forward primer (SEQ ID NO: 25):
5′-TTATTTAGGAAAAATAGAGAAAGGGA-3 ′ (5 ′ end is biotinylated)
Reverse primer for PCR (SEQ ID NO: 26):
5'- CCTAACAAACTTTCCTACCCTAAAC-3 '
Template DNA: Human genomic DNA 1 μg
Nucleotides: dATP, dGTP, and dTTP are each 2.5 μmole, and 2.5 μmole dCTP and 0.5 μmole 5-methyl dCTP (total addition amount of dCTP and 5-methyl dCTP is 2.5 μmole) are used.
Total amount of PCR reaction system: 50 μL
PCR reaction conditions:
(95 ° C, 2 minutes) x 1 cycle (95 ° C, 30 seconds / 59 ° C, 30 seconds / 72 ° C, 30 seconds) x 30 cycles (72 ° C, 8 minutes) x 1 cycle

  The DNA product obtained by PCR amplification was diluted to 100 μL by diluting 5 μL with 10 mmol / L Tris-HCl buffer (pH 8.0) / 1 mmol / L EDTA (hereinafter referred to as TE buffer). The total amount of the solution was added to a 96-well ELISA plate and allowed to react at room temperature for 1 hour. As the ELISA plate, each well was previously coated with avidin and further blocked with 10% bovine serum albumin. After the reaction, each well was washed several times with TE buffer, and the amount of DNA coated on the well was quantified by a fluorometric method using Pico Green (Cat # P7581; manufactured by Molecular Probes, USA) according to the manual.

Next, each well was treated with 0.1 mol / L hydrochloric acid for 5 minutes to denature the DNA immobilized on the well to form a single strand, and then 0.1% Tween20 / PBS (pH 7.5) / 1 mmol / Washed several times with L EDTA (hereinafter referred to as T-EPBS). By this operation, only the biotinylated sense strand DNA remained on the well, and the non-biotinylated antisense strand was washed away.
When the above operation is performed, a DNA product in which 5-methyl dCTP is randomly incorporated into about 20% of the cytosine of the sense strand in the genomic DNA to be examined is located at about 20% of the cytosine methylated at position 5. Only the sense strand of the DNA artificially incorporated with 5-methyl dCTP remains on the avidin-coated ELISA plate and is bound thereto.

  Next, an anti-5-methylcytosine antibody (mouse monoclonal antibody) adjusted to a concentration of 2 to 5 μg / mL using T-EPBS was placed in each well and incubated for 1 hour. After the reaction, each well was washed 4 to 5 times with T-EPBS, and peroxidase-labeled anti-mouse IgG diluted 2000 times with T-EPBS was placed in each well and incubated for 1 hour. After the reaction, each well was washed 5 to 6 times with T-EPBS, peroxidase substrate 100 μL (Cat. # T-0440; SIGMA, USA) was added, and incubated at room temperature for 30 minutes, 0.5 mol / L sulfuric acid 20 μL. Was added to stop the reaction, and the absorbance at a wavelength of 450 nm was measured.

The preparation and measurement of the methylated standard DNA is a DNA having a chain length approximate to the DNA to be measured, and a PCR product that does not contain 5-methylcytosine in the DNA is used as a 0% methylated standard DNA. The DNA product obtained by mixing cytosine: 5-methylcytosine at a ratio of 95: 5 at the time of amplification was used as a 5% methylated standard DNA, and the ELISA plate was coated in the same manner as the human genomic DNA sample to be examined. Thereafter, the same operation was performed.
Absorbance was carried out using E-max manufactured by Molecular Devices. Further, SPECTRAmax GEMINI XS manufactured by Molecular Devices was used as the fluorometer, and the fluorescence intensity was measured at an excitation wavelength of 480 nm and a fluorescence wavelength of 520 nm. The results are shown in Table 6. In addition, “human genomic DNA sample 1” and “human genomic DNA sample 2” in Table 6 are the same DNA sample, but differ only in the dilution rate.

<< Table 6 >>
Relative fluorescence intensity Absorbance (Ab) Ab / Fr
(Fr) (450 nm)
0% methylated standard DNA 1.000 0.085 0.085
5% methylated standard DNA 0.824 0.306 0.371
Human genomic DNA sample 1 0.291 0.106 0.364
Human genomic DNA sample 2 1.205 0.374 0.310

When the reaction efficiency of the hydrogen sulfite ion treatment is high, an approximate value of the methylation rate of the DNA to be examined can be obtained by the above method. On the other hand, in the case of a DNA sample with low reaction efficiency of bisulfite ion treatment, cytosine in the DNA to be tested does not completely change to uracil, so that unmethylated cytosine in the DNA to be tested is directly PCR amplified. Therefore, even if the DNA to be examined is not methylated at all, it is measured as if it is apparently methylated. Therefore, when the reaction efficiency of the bisulfite ion treatment is low, the bisulfite ion treatment is performed using DNA in which 5-methylcytosine is incorporated in the DNA to be examined at an arbitrary ratio as a starting material, and this is treated with the methyl of the DNA to be examined. The methylation rate can be accurately quantified by comparing with the conversion rate.
Here, methylated standard DNA is prepared, and the methylation of the sample is calculated as a standard substance that becomes methylated standard DNA as it is. To measure the methylation rate of the sample DNA more accurately, A more preferable quantification method can be obtained by subjecting the artificially incorporated DNA to 5-methylcytosine by the PCR method and treating with bisulfite ion and comparing the methylation rate with the methylation rate of the DNA to be measured.

Example 3: Staining of metaphase chromosome specimen of cell division
In this example, as normal cells, lung fibroblast-derived TIG-1 cells [National Pharmaceutical Food Sanitation Research Institute, JCRB (Japanese Collection of Research Bioresources) cell bank] were used, and cancer cells were Raji cells derived from Burkitt lymphoma [Tohoku University Institute for Aging Medicine, Medical Cell Resource Center; Catalog No. TKG0371] and HT1080 cells derived from fibrosarcoma [Tohoku University Institute for Aging Medicine, Medical Cell Resource Center; Catalog No. TKG0202] was used.

Demecorsin was added to the cells cultured in the culture dish so that the final concentration in the medium was 0.02 μg / mL, and the cells were further cultured for 30 minutes.
For Raji cells, which are floating cells, the cultured cells were removed from the culture dish together with the medium, centrifuged at 1000 rpm for 2 minutes, and the supernatant was discarded to recover the cells.
On the other hand, for HT1080 cells and TIG-1 cells, which are adherent cells, the cells on the culture dish are lightly rinsed with Hanks solution (without magnesium and calcium), and the Hanks solution containing 0.25% trypsin is cultured at 75 cm ² . Add 2 mL to the dish, dispose after spreading over the whole cell, incubate at 37 ° C. for 2 minutes, collect the detached cells with 5 mL of medium, centrifuge at 1000 rpm for 2 minutes, and discard the supernatant. Cells were collected.

To the pelleted cells, 5 mL of 75 mmol / L potassium chloride solution was added to sufficiently suspend the cells, and left at room temperature for 20 minutes. Next, after the cell suspension was iced, 1 mL of Carnoy's solution (methanol: acetic acid = 3: 1) was added over 15 minutes while mixing well, and then 4 mL of the Carnoy's solution was added over 5 minutes. Next, the cell suspension was centrifuged at 1500 rpm for 5 minutes at 4 ° C., and the supernatant was discarded. The precipitated cells were suspended in the Carnoy's solution and stored at −20 ° C. until use.
A slide glass thoroughly washed with 99.5% ethanol was placed at a position 3 cm from the surface of a water bath kept at 60 ° C., and after the slide glass was sufficiently warmed, the Carnoy's solution was introduced from a position 20 cm above the slide. One drop of the cell suspension suspended in the solution was dropped, removed about 30 seconds later, excess water on the slide was removed with a filter paper, left at room temperature for 2 to 10 days, and the following chromosome staining was performed.

  A chromosome sample (slide glass) was treated with ice-cooled 10 mmol / L phosphate buffer (pH 7.6) (hereinafter referred to as PB) for 2 minutes, and then the sample was placed on ice and cooled on ice. Treated with 0.05% trypsin / 10 mmol / L phosphate buffer (pH 7.6) for 1 minute. Next, it was treated with ice-cooled 70% ethanol and 99.5% ethanol for 2 minutes in order, completely air-dried, and then treated with 2N HCl at room temperature for 10 minutes. Then, it was treated with 0.1 mol / L Tris-HCl (pH 8.0) at room temperature for 10 minutes. This was lightly rinsed with PB containing 0.15 mol / L NaCl (hereinafter referred to as PBS) and again treated with 70% ethanol and 99.5% ethanol for 2 minutes successively. Thereafter, the specimen was dried at room temperature.

  After completely drying, 1% bovine serum albumin (hereinafter referred to as BSA) / PBS is added with an avidin blocking solution (Avidin-Biotin blocking kit, Cat. # SP-2001; Vector) to obtain a blocking solution. This was placed on the specimen and reacted at room temperature for 30 minutes. Next, the specimen was washed with PBS containing 0.05% Tween-20 (hereinafter referred to as PBS-T) and further subjected to the following primary antibody reaction. A biotin blocking solution (Avidin-Biotin blocking Kit, Cat. # SP) was added to an anti-5-methylcytosine monoclonal antibody (primary antibody) with an antibody diluent (0.5% BSA / PBS-T) to a final concentration of 20 μg / mL. -2001; Vector) was added to obtain a primary antibody solution. This was placed on the specimen, reacted at room temperature for 1 hour, and then washed with PBS-T. About half of the chromosome samples (150 mmol / L NaCl treatment group), the following secondary antibody reaction was carried out as it was, and for the remaining half (500 mmol / L NaCl treatment group), high salt concentration PBS-T [10 mmol / L phosphate buffer (pH 7.5) /0.5 mol / L NaCl / 0.05% Tween-20] was further washed for 5 minutes, and then the secondary antibody reaction was carried out. As the secondary antibody solution, an anti-mouse biotinylated secondary antibody (Cat. # E0354; Cappel) diluted with 1% BSA / PBS-T 800 times was used. This was placed on the specimen and allowed to react for 30 minutes at room temperature. After thoroughly washing with PBS-T, avidin-FITC solution (Cat. # A-2001; Vector) diluted 10,000-fold with 1% BSA / PBS-T is placed on the specimen and reacted at room temperature for 30 minutes. I let you. After thoroughly washing with PBS-T, the washing solution was blotted with water-absorbing paper and encapsulated using an encapsulant (Cat. # H-1000; Vector). The stained specimen was observed with a fluorescence microscope (Cat. # TE2000-U; Nikon) and photographed with a digital camera.

The results are shown in FIGS. 11 to 13 are a 150 mmol / L NaCl treatment group, and FIGS. 14 to 17 are a 500 mmol / L NaCl treatment group. 11 and 14 are Raji cells that are cancer cells, FIGS. 12 and 15 are HT1080 cells that are cancer cells, and FIGS. 13, 16, and 17 are TIG-1 cells that are normal cells. It is.
In the usual immunostaining, washing is performed by reacting an antibody under physiological conditions using PBS (Nacl concentration = 0.15 mol / L) or the like. Under such general immunostaining conditions, no difference was observed in chromosome staining images of HT1080 cells (FIG. 12) as cancer cells and TIG-1 cells (FIG. 13) as normal cells. For Raji cells (FIG. 11), which are cancer cells, intense staining spots were observed in individual chromosomes in the staining under such physiological conditions. Therefore, under the commonly used immunostaining conditions, abnormal methylation of chromosomes in Raji cells was observed, although the difference from the methylation of chromosomes in TIG-1 cells was small. However, with regard to HT1080 cells, the difference in chromosome methylation from normal cells was not observed under the general immunostaining conditions.

  On the other hand, after the primary antibody reaction, when washed with a buffer solution having a higher salt concentration (NaCl concentration = 500 mmol / L) than under physiological conditions, observation should be made under physiological staining conditions as shown in FIGS. It was possible to observe the difference in chromosomal methylation between normal cells (TIG-1 cells) and cancer cells (HT1080, Raji cells) that could not be detected. It is considered that the antibody that weakly bound to the chromosome sample (monovalent binding antibody) was removed by the washing, and the strongly bound antibody (bivalent binding antibody) remained. As a result of staining the chromosome subjected to the high salt concentration treatment, fine spots (spots) that are positive staining sites are fine in cancer cells (FIGS. 14 and 15), and spots in normal cells (FIGS. 16 and 17). Largely observed. In addition, the number of spots in cancer cells (FIGS. 14 and 15) is larger than the number of spots in normal cells (FIGS. 16 and 17), and normal cells and cancer cells can be easily distinguished. There was found.

<< Reference Example 1: Determination of divalent bonding conditions >>
In this reference example, the bivalent binding conditions of the anti-5-methylcytosine monoclonal antibody used as the primary antibody in Example 3 were determined.
First, a Fab fragment is prepared from an anti-5-methylcytosine monoclonal antibody (IgG2a, κ) according to a conventional method, and this Fab fragment (monovalent) and whole IgG molecule (divalent) are used at various NaCl concentrations. The binding ability was evaluated by ELISA. As an antigen to be immobilized on an ELISA plate, double-stranded DNA in which 5-methylcytosine was randomly incorporated by PCR using a reverse primer and a biotinylated forward primer was used, and biotin was introduced into each well coated with avidin. After fixing the double-stranded DNA, it was used in a single-stranded state by treatment with 50 mmol / L hydrochloric acid. As a secondary antibody, horseradish peroxidase (HRP) labeled anti-mouse IgG antibody was used, and paranitrophenyl phosphate was used as a chromogenic substrate, and absorbance at 405 nm was measured.

  The results are shown in FIG. As shown in FIG. 18, the Fab fragment (monovalent) rapidly decreases in binding capacity as the NaCl concentration increases. For example, at a concentration of 0.3 mol / L, the maximum binding capacity (at a concentration of 0.05 mol / L). At a concentration of 0.5 mol / L, the maximum binding capacity was reduced to 10% or less. On the other hand, the whole IgG molecule (divalent) has a moderate decrease in binding ability compared to the Fab fragment, for example, about 50% of the maximum binding ability at a concentration of 0.5 mol / L, and a concentration of 0.8 mol / L. The binding ability of about 25% was maintained. From the graph shown in FIG. 18, the divalent binding condition of this anti-5-methylcytosine monoclonal antibody is 0.3 to 1.0 mol / L, preferably 0.4 to 0.8 mol / L, more preferably 0.5. It could be determined to be ~ 0.6 mol / L.

  The measurement method and evaluation method of the present invention can be applied to uses such as evaluation of malignancy of cancer cells or evaluation of the differentiation state of various cells.

  The numerical heading <223> in the sequence listing below describes the “Artificial Sequence”. Specifically, each base sequence represented by SEQ ID NOs: 2 and 4 is a sequence after bisulfite ion treatment, and each base sequence represented by SEQ ID NOs: 5 and 10 is a sequence after PCR amplification. The base sequences represented by SEQ ID NOs: 6 and 8 are forward primers, the base sequences represented by SEQ ID NOs: 7 and 9 are reverse primers, and the base sequences represented by SEQ ID NO: 11 are It is a sequence before bisulfite ion treatment, and each base sequence represented by SEQ ID NOs: 12, 13, and 15 is a sequence after bisulfite ion treatment and PCR amplification, and the base sequence represented by SEQ ID NO: 16 is 1 is the sequence (a) in FIG. 1, the base sequence represented by SEQ ID NO: 17 is the sequence (b) in FIG. 1, and the base sequence represented by SEQ ID NO: 18 is the sequence (c) in FIG. And SEQ ID NO: 19 The base sequence represented is the sequence (d) in FIG. 1, the base sequence represented by SEQ ID NO: 20 is the sequence (e) in FIG. 2, and the base sequence represented by SEQ ID NO: 21 is 2, the base sequence represented by SEQ ID NO: 22 is the sequence (g) in FIG. 2, and the base sequence represented by SEQ ID NO: 23 is the sequence (h) in FIG. The base sequence represented by SEQ ID NO: 25 is a forward primer, and the base sequence represented by SEQ ID NO: 26 is a reverse primer.

It is explanatory drawing which shows typically each process of the double strand separation-sense strand template-C type | mold measuring method and double strand separation-sense strand template-G type | mold measuring method of this invention. It is explanatory drawing which shows typically each process of the double strand separation-antisense strand template-C type | mold measuring method and double strand separation-antisense strand template-G type | mold measuring method of this invention. It is explanatory drawing which shows typically the area | region which can be made into a measuring object base sequence in this invention method. It is the photograph which replaces drawing which shows the result of the electrophoresis obtained by implementing the method of this invention using the methylated DNA reference standard whose methylation rate is 25%. It is a graph which shows the relationship between the methylation rate of cytosine at the time of using FITC label | marker dCTP, and a fluorescence ratio (sense strand / antisense strand). It is a graph which shows the relationship between the methylation rate of cytosine and the fluorescence ratio (antisense strand / sense strand) when Texas Red labeled dGTP is used. It is a schematic diagram which shows the state of the monovalent | monohydric coupling | bonding with which single molecule antibody couple | bonds with double stranded DNA in one antigen binding site. It is a schematic diagram which shows the state of the bivalent coupling | bonding in which the molecule | numerator antibody couple | bonds with double stranded DNA in two antigen binding sites. FIG. 3 is a schematic diagram showing a bivalent binding state in which bimolecular antibodies fixed to a carrier bind to double-stranded DNA at each antigen binding site. It is a schematic diagram which shows the state which the bimolecular antibody has couple | bonded with double stranded DNA in each one antigen binding site. It is the microscope picture which replaces drawing which shows the chromosome dyeing | staining image of the Raji cell (150 mmol / L NaCl processing group) which is a cancer cell. It is the microscope picture which replaces drawing which shows the chromosome dyeing | staining image of HT1080 cell (150 mmol / L NaCl processing group) which is a cancer cell. It is the microscope picture which replaces drawing which shows the chromosome dyeing | staining image of the TIG-1 cell (150 mmol / L NaCl processing group) which is a normal cell. It is the microscope picture which replaces drawing which shows the chromosome dyeing | staining image of the Raji cell (500 mmol / L NaCl processing group) which is a cancer cell. It is the microscope picture which replaces drawing which shows the chromosome dyeing | staining image of HT1080 cell (500 mmol / L NaCl processing group) which is a cancer cell. It is the microscope picture which replaces drawing which shows the chromosome dyeing | staining image of the TIG-1 cell (500 mmol / L NaCl processing group) which is a normal cell. It is the microscope picture which replaces drawing which shows the chromosome dyeing | staining image of the TIG-1 cell (500 mmol / L NaCl processing group) which is a normal cell. It is a graph which shows the binding ability of an anti- 5-methylcytosine monoclonal antibody (IgG) and its Fab fragment in various concentrations of NaCl conditions.

Claims (5)

(1) a step of treating single-stranded or double-stranded DNA containing a base sequence to be measured with a cytosine modifying agent capable of converting cytosine into another base;
(2) Labeled deoxycytidine 5′-triphosphate or labeled deoxyguanosine 5′-trilin using one of the obtained double-stranded DNAs or the obtained single-stranded DNA as a template A step of performing a DNA amplification operation in the presence of an acid; and (3) the amount of labeled cytosine or labeled guanine in the amplified double-stranded DNA, or in the amplified double-stranded DNA. A method for measuring a cytosine methylation rate in a base sequence to be measured, which comprises the step of measuring (1) a step of treating single-stranded or double-stranded DNA containing a base sequence to be measured with a cytosine modifying agent capable of converting cytosine into another base;
(2) Labeled deoxycytidine 5′-trilin using the obtained double-stranded DNA as a template, the DNA strand containing the converted base sequence derived from the base sequence to be measured, or the obtained single-stranded DNA A step of performing a DNA amplification operation in the presence of an acid, and (3) a DNA strand containing an amplified base sequence corresponding to the post-conversion base sequence of the amplified double-stranded DNA, or an amplified double-stranded DNA The measurement method according to claim 1, further comprising a step of measuring the amount of labeled cytosine therein. (1) a step of treating single-stranded or double-stranded DNA containing a base sequence to be measured with a cytosine modifying agent capable of converting cytosine into another base;
(2) A labeled deoxyguanosine 5′-trilin using the obtained double-stranded DNA containing a converted DNA sequence derived from the base sequence to be measured or the obtained single-stranded DNA as a template A step of performing a DNA amplification operation in the presence of an acid; and (3) a complementary strand of an amplified double-stranded DNA containing a post-amplification base sequence corresponding to the post-conversion base sequence, or an amplified double strand The measurement method according to claim 1, further comprising a step of measuring the amount of labeled guanine in the double-stranded DNA. (1) a step of treating single-stranded or double-stranded DNA containing a base sequence to be measured with a cytosine modifying agent capable of converting cytosine into another base;
(2) In the presence of labeled deoxycytidine 5′-triphosphate using, as a template, a complementary strand of the obtained double-stranded DNA containing a converted base sequence derived from the base sequence to be measured, A step of performing a DNA amplification operation, and (3) a complementary strand of a DNA strand containing an amplified base sequence corresponding to the base sequence after conversion of the amplified double-stranded DNA, or a label in the amplified double-stranded DNA The measurement method according to claim 1, comprising a step of measuring the amount of oxycytosine. (1) a step of treating single-stranded or double-stranded DNA containing a base sequence to be measured with a cytosine modifying agent capable of converting cytosine into another base;
(2) In the presence of labeled deoxyguanosine 5′-triphosphate, using as a template the complementary strand of the DNA strand containing the converted base sequence derived from the base sequence to be measured of the obtained double-stranded DNA, A step of performing a DNA amplification operation; and (3) a DNA strand containing an amplified base sequence corresponding to the base sequence after conversion of the amplified double-stranded DNA, or labeled guanine in the amplified double-stranded DNA. The measuring method according to claim 1, further comprising a step of measuring the amount.

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