JP2006149334A - Method for detecting methylation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect even a trace amount of a methylated DNA contained in a sample DNA with high sensitivity. <P>SOLUTION: The method for detecting methylation comprises a step A of treating a DNA with a methylation-sensitive enzyme, a step B of carrying out an amplification reaction for amplifying a region containing a site of the object of inspection in the DNA after the step A and a step C of detecting a DNA fragment obtained by the amplification reaction after the step B. The presence or absence of the methylation in th site of the object of inspection is judged on the basis of the results of detection of the DNA fragment. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a methylation detection method for detecting the presence or absence of methylation in DNA, and more particularly, to a methylation detection method that can be used for detection of diseases caused by the presence or absence of methylation.

  The genome is a blueprint of an organism, and in humans it is made from deoxyribonucleic acid (DNA) of about 3 billion base pairs consisting of a 4-base combination of A (adenine) T (thymine) G (guanine) C (cytosine). Yes. Some of the constituent bases of these genomic DNAs are methylated. The presence or absence of this methylation has been found to play a very important role as genetic information in the genome and as part of the transcriptional regulatory mechanism. In many animals including humans, C of the base sequence CG may be methylated. The presence or absence of methylation is basically inherited even after cell division, and the methylated region is basically It is known that transcription is inactivated.

  Genomic DNA methylation occurs at multiple locations, and the methylation pattern varies depending on the differentiation process, cell type, cancer cell, and the like. Examining the methylation state of a specific DNA region having a correlation between the presence or absence of methylation and a disease state such as the cell type and the degree of progression of cancer is very important information. By examining the presence or absence of methylation at multiple sites, it may be possible to diagnose the differentiation state of cells and the progression of cancer, and it is necessary to be able to test efficiently and accurately even when there are many samples at multiple locations per genome. .

  Various methods for examining the methylation status of the genome have been developed. Currently, there are two typical principles, a method of judging by digestion of genomic DNA with a methylation-sensitive restriction enzyme, and genomic DNA with sodium bisulfite. There is a method (Patent Document 1) for analyzing the presence or absence of deamination by a sequence or the like. As a method using a methylation-sensitive restriction enzyme, there is a method of measuring the length of a target fragment by Southern blotting. In the method using sodium bisulfite, there is a method of examining whether the amplification reaction occurs using a sequence of a target region or a primer containing a methylation site.

  In addition, a method for examining the methylation state of a genome using a microarray is also known (Patent Documents 2 and 3).

International Publication Number WO2002 / 031186 JP 2003-38183 A JP 2003-144172 A

  However, the method of measuring the length of the target fragment by Southern blotting using a methylation-sensitive restriction enzyme has the advantage that the ratio of methylation and unmethylation can be inferred from the density of the band, but a small amount of sample DNA There is a problem that detection using is difficult.

  On the other hand, in the method using sodium bisulfite, there is an advantage that the methylation state can be comprehensively examined in any nucleic acid sequence without depending on the restriction enzyme recognition sequence, but the operation of the sequence is complicated, There is a problem that processing when there are many samples is difficult.

  Accordingly, in view of the actual situation as described above, an object of the present invention is to provide an excellent methylation detection method that can detect with high sensitivity even if the amount of methylated DNA contained in a sample DNA is very small. .

The present invention that has achieved the above-described object includes the following.
(1) Step A of treating DNA with a methylation-sensitive enzyme;
After the step A, a step B for performing an amplification reaction for amplifying a region containing the site to be examined in the DNA,
After step B, including step C for detecting a DNA fragment obtained by the amplification reaction,
A methylation detection method characterized by determining the presence or absence of methylation at the site to be inspected based on a detection result of a DNA fragment.

  (2) The method for detecting methylation according to (1), wherein the step B uses at least one or more primer sets designed to sandwich the site to be examined.

  (3) The method for detecting methylation according to (1), wherein in the step B, a plurality of regions including the site to be examined are amplified so as to have different DNA fragment lengths.

  (4) The method for detecting methylation according to (1), wherein in the step C, the length of the DNA fragment is detected by electrophoresis.

(5) In the above step B, a region containing no methylation sensitive enzyme recognition sequence is amplified as a positive control DNA fragment,
In the step C, the progress of the amplification reaction in the step B is verified by detecting the positive control DNA fragment, The methylation detection method according to (1),

(6) In the above step A, a methylation-sensitive enzyme recognition sequence that is methylated and / or a control DNA fragment containing a methylation-sensitive enzyme recognition sequence that is not methylated is treated with a methylation-sensitive enzyme,
In the step B, an amplification reaction for amplifying a region containing a methylation sensitive enzyme recognition sequence contained in the control DNA fragment is performed,
In the step C, the methylation detection method according to (1), wherein the progress of the enzymatic reaction by the methylation sensitive enzyme is verified by detecting an amplified fragment using the control DNA fragment as a template.

  (7) The methylation detection method according to (1), wherein, in the step C, an amplified fragment obtained by performing the step B without performing the step A is used as a marker.

(8) In step B, an amplification reaction is performed using a primer set including a primer having a labeling substance,
In the step C, the DNA fragment obtained by the amplification reaction in the step B is detected by detecting a labeling substance, The methylation detection method according to (1),

  (9) The methylation detection method according to (8), wherein the labeling substance is a fluorescent substance that exhibits a different fluorescence wavelength for each primer set.

  ADVANTAGE OF THE INVENTION According to this invention, regarding the presence or absence of methylation in a test object site | part, even if there are multiple test object site | parts, the methylation detection method which can obtain the stable result efficiently can be provided.

  Hereinafter, the methylation detection method according to the present invention will be described in detail with reference to the drawings. The methylation detection method according to the present invention is a method capable of detecting the presence or absence of methylation at a predetermined site in DNA.

  The methylation detection method according to the present invention includes a step A in which DNA is treated with a methylation-sensitive enzyme, and a step B in which an amplification reaction for amplifying a region including the site to be examined in the DNA is performed after the step A. And after step B, a step C of detecting the DNA fragment obtained by the amplification reaction.

  In step A, DNA containing a methylation test target site is first treated with a methylation sensitive enzyme. Here, the methylation detection target site is a base included in a sequence (hereinafter referred to as a methylation sensitive enzyme recognition sequence) that is recognized and cleaved by a methylation sensitive enzyme described later, and may be methylated. Means a base having The methylation-sensitive enzyme means an enzyme that cannot cleave DNA when a predetermined base in the base sequence recognized by the enzyme is methylated and cleaves DNA when the base is not methylated. Examples of methylation sensitive restriction enzymes include HapII, NotI, SfiI, FseI, CspI, MluI, AscI, BssHII, ClaI, XhoI, Eco521, SalI, NarI, NruI, PsrII, PacI, SwaI, SseB387I, PmeI and the like. However, it is not limited to these.

  The treatment of DNA with a methylation sensitive enzyme means that at least one of the above methylation sensitive enzymes is added to a DNA solution prepared at a predetermined temperature, a predetermined pH, and a predetermined salt concentration to cause a DNA cleavage reaction to proceed. means. Here, various conditions such as temperature, pH, and salt concentration vary depending on the methylation sensitive enzyme used, and can be appropriately determined according to the methylation sensitive enzyme used.

  Next, in step B, an amplification reaction is performed to amplify a region including the methylation test target site. Here, as an example of the amplification reaction, a PCR (polymerase chain reaction) method can be cited, but the DNA subjected to methylation-sensitive enzyme treatment in step A is used as a template and includes a site for methylation test. Any method that can amplify the region may be used. Such amplification reactions include LCR (ligase chain reaction), gap LCR, SDA (strand displacement amplification), Qβ replicative amplification, TAS (transcription amplification system), 3SR (self-sustained sequence relication system) , NASBA (nucleic acid sequence-based amplification) method, TMA (transcription-mediated amplification) method, ICAN (isothermal and chimeric primer-initiated amplification of nucleic acid) method, LAMP (loop-mediated isothermal amplification) method .

  When the PCR method is applied as an amplification reaction, the pair of primers used is designed so as to sandwich the site to be examined. Further, in the methylation detection method according to the present invention, when a plurality of examination target sites are used, a pair of primers is designed for each of a plurality of regions including one test target site. Various conditions such as the temperature cycle and reaction solution composition in the PCR reaction can be appropriately determined according to, for example, the designed Tm value of the primer, the number of amplification regions, the amplification site, and the like.

  In addition, when a plurality of regions including one site to be examined are amplified simultaneously, it is preferable to design primers so that the amplified fragments have different lengths. This is because by designing the primers so that the amplified fragments have different lengths, the presence or absence of the amplified fragments can be easily determined in Step C described later.

  Furthermore, the primer used for the amplification reaction may be one to which a fluorescent label is bound. By using a primer to which a fluorescent label is bound, the amplified fragment can be more easily detected in Step C described later. When a pair of primers is used for the amplification reaction, the fluorescent label may be bound to at least one primer. When a plurality of pairs of primers are used for the amplification reaction, it is preferable to bind a fluorescent label showing a different fluorescence wavelength for each pair.

  In step B, the amplification reaction does not proceed when the DNA is cleaved by the methylation sensitive enzyme in step A, but the amplification reaction does not proceed when the DNA is not cleaved by the methylation sensitive enzyme in step A. proceed. Therefore, by detecting the progress of the amplification reaction in step B, it is possible to detect the presence or absence of methylation at the methylation detection target site contained in the DNA used in step A.

  That is, in step C, the DNA fragment obtained by the amplification reaction is detected. The technique for detecting the DNA fragment is not limited in any way. For example, it is preferable to apply an electrophoresis method using the reaction solution after the amplification reaction in Step B as it is. Further, in Step C, the method of detecting fluorescence of a fluorescent label bound to a primer can be applied without being limited to the detection of DNA fragments by applying electrophoresis.

  As a result of Step C, when amplification of a DNA fragment containing a methylation detection target site is confirmed, it can be determined that the methylation detection target site is methylated. Conversely, when amplification of a DNA fragment containing a methylation detection target site is not confirmed as a result of step C, it can be determined that the methylation detection target site is not methylated.

  According to the methylation detection method including the steps A to C described above, an efficient and stable result can be obtained regarding the presence or absence of methylation in the site to be examined, even if there are a plurality of sites to be examined. In addition, in the DNA prepared in step A, methylation can be reliably detected in the methylation detection target site even when the methylation detection target site is a mixture of methylated DNA and non-methylated DNA. can do.

  In the methylation detection method described above, it is preferable to amplify a region that does not contain a methylation-sensitive enzyme recognition sequence in step B as a positive control DNA fragment. That is, the region that does not include the methylation-sensitive enzyme recognition sequence is a region that is reliably amplified in step B because it is not cleaved by the methylation-sensitive enzyme in step A regardless of the presence or absence of methylation. Therefore, in this case, it is possible to verify the progress of the amplification reaction in Step B by detecting the positive control DNA fragment in Step C.

  Furthermore, in the methylation detection method described above, in step A, in addition to the DNA containing the site to be examined, a control DNA fragment containing a methylated methylation sensitive enzyme recognition sequence is similarly treated with a methylation sensitive enzyme. In the amplification reaction of the above step B, it is preferable to amplify a region containing a methylation sensitive enzyme recognition sequence contained in the control DNA fragment. In this case, the control DNA fragment is not cleaved in the step A, and the region is surely amplified by the step B. In this case, in Step C, an amplification product derived from the control DNA fragment can be detected. Therefore, by detecting the amplification product derived from the control DNA fragment, it is possible to verify the progress of the enzyme reaction by the methylation sensitive enzyme.

  In this case, in step A, in addition to the DNA containing the site to be examined and the control DNA fragment, other control DNA fragments containing a methylated methylation sensitive enzyme recognition sequence are similarly treated with a methylation sensitive enzyme. In the amplification reaction of Step B, it is preferable to amplify a region containing a methylation sensitive enzyme recognition sequence contained in the other control DNA fragment. In this case, since the other control DNA fragments are cleaved in step A, the region is not amplified in step B, and in step C, amplification products derived from other control DNA fragments are not detected. Therefore, it is possible to verify the progress of the enzymatic reaction by the methylation sensitive enzyme by confirming that there is no amplification product derived from the other control DNA fragment. In this case, false positive and weak positive can be reliably determined for methylation of the methylation detection target site.

  Furthermore, in the methylation detection method described above, in step B, in addition to the DNA treated with the methylation sensitive restriction enzyme in step A, DNA comprising the same base sequence not treated with the methylation sensitive restriction enzyme is used. It is preferable to carry out an amplification reaction. In this case, the amplified fragment derived from DNA that has not been treated with a methylation sensitive restriction enzyme can be used as a marker for the amplified fragment derived from DNA treated with the methylation sensitive restriction enzyme in Step A. Thus, for example, when detecting by electrophoresis in step C, by performing electrophoresis in a different lane after performing step B using DNA not treated with a methylation sensitive restriction enzyme, The migration pattern can be used as a migration pattern for the marker.

  Hereinafter, the methylation detection method according to the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to the following examples.

[Example 1]
FIG. 1 is a schematic diagram when a genomic DNA is treated with a methylation-sensitive restriction enzyme and then a PCR reaction is performed with a restriction enzyme recognition site in between. Here, HapII was used as a methylation sensitive restriction enzyme. As shown in the figure, when the restriction enzyme recognition site is methylated, DNA cannot be cleaved by a methylation-sensitive restriction enzyme, so that an amplification product is obtained by performing a PCR reaction. On the contrary, when the restriction enzyme recognition site is not methylated, DNA can be cleaved by a methylation sensitive restriction enzyme, so that when PCR reaction is performed, an amplification product cannot be obtained.

  FIG. 2 is a flow of a specific experiment of the methylation detection method of this example. First, a methylated control DNA having a known sequence serving as a positive control and an unmethylated control DNA having a known sequence serving as a negative control are added to the genomic DNA serving as a sample. Next, these DNA mixed solutions are treated with a methylation sensitive restriction enzyme. A PCR reaction is performed using a plurality of primer sets, using the enzyme-treated DNA mixed solution and the untreated DNA mixed solution of only one sample as a template. Here, a total of 10 primer sets including a primer set (8 sets) designed with 8 restriction enzyme recognition sites from a to h and a positive control and negative control primer set were used. If methylated DNA is not available as a positive control template, the positive control primer should be designed so that it does not intervene restriction enzyme recognition sites. Finally, each amplified product is electrophoresed to analyze the presence or absence of methylation based on the presence or absence of a band.

  FIG. 3 is an electrophoretogram of the amplification product obtained in FIG. Lane 1 uses a DNA mixed solution that has not been treated with a methylation-sensitive restriction enzyme as a template. Since the template DNA is not cleaved at all, the 10 primer sets are all amplified and 10 bands are seen. Lanes 2-6 contain unknown sample genomes. Since there is no positive control band in Lane 5, it is highly likely that the specificity was cleaved with a methylation-sensitive restriction enzyme treatment, or amplification in the PCR reaction was incomplete. Similarly, lane 6 is a failed sample because there is a negative control band and the methylation sensitive restriction enzyme treatment is likely to be incomplete. Lane 2 is methylated at sites b, c, e, and h, Lane 3 is methylated at sites a, c, d, and g, and Lane 4 is methylated at any site. You can see that it is not.

Schematic diagram when a genomic DNA is treated with a methylation sensitive restriction enzyme and then subjected to a PCR reaction with a restriction enzyme recognition site in between. The flowchart which shows the flow of the concrete experiment of the methylation detection method. Electrophoresis diagram of amplification product.

Claims (9)

Treating DNA with a methylation sensitive enzyme; and
After the step A, a step B for performing an amplification reaction for amplifying a region containing the site to be examined in the DNA,
After step B, including step C for detecting a DNA fragment obtained by the amplification reaction,
A methylation detection method characterized by determining the presence or absence of methylation at the site to be inspected based on a detection result of a DNA fragment.   The method for detecting methylation according to claim 1, wherein the step B uses at least one primer set designed to sandwich the site to be examined.   The method for detecting methylation according to claim 1, wherein in step B, a plurality of regions including the site to be examined are amplified so as to have different DNA fragment lengths.   The method for detecting methylation according to claim 1, wherein in the step C, the length of the DNA fragment is detected by electrophoresis. In the above step B, a region containing no methylation sensitive enzyme recognition sequence is amplified as a positive control DNA fragment,
The method for detecting methylation according to claim 1, wherein in the step C, the progress of the amplification reaction in the step B is verified by detecting the positive control DNA fragment. In the above step A, a control DNA fragment containing a methylated methylation sensitive enzyme recognition sequence and / or an unmethylated methylation sensitive enzyme recognition sequence is treated with a methylation sensitive enzyme,
In the step B, an amplification reaction for amplifying a region containing a methylation sensitive enzyme recognition sequence contained in the control DNA fragment is performed,
The method for detecting methylation according to claim 1, wherein in the step C, the progress of the enzymatic reaction by the methylation sensitive enzyme is verified by detecting an amplified fragment using the control DNA fragment as a template.   The method for detecting methylation according to claim 1, wherein, in the step C, an amplified fragment obtained by performing the step B without performing the step A is used as a marker. In the above step B, an amplification reaction is performed using a primer set including a primer having a labeling substance,
The method for detecting methylation according to claim 1, wherein in the step C, a DNA fragment obtained by the amplification reaction in the step B is detected by detecting a labeling substance.   9. The method for detecting methylation according to claim 8, wherein the labeling substance is a fluorescent substance having a different fluorescence wavelength for each primer set.

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