JP2003038183A - Biochip for detecting methylation of methylation site in genomic DNA and method for detecting methylation - Google Patents

Abstract

(57) [Summary] [Problem] To comprehensively analyze methylation sites in a genome. SOLUTION: Among the methylation-sensitive restriction enzyme SmaI and the methylation-insensitive restriction enzyme XmaI that cleave the same recognition site, Sm
Genomic DNA 700 digested with aI and genomic DNA digested with SmaI
Digest NA with XmaI. DNs whose ends are XmaI cuts
The A fragment 720 is specifically amplified, and the amplified DNA fragment is detected using a biochip having a corresponding probe immobilized on a substrate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting methylation at a methylation site in genomic DNA and a biochip used for the method.

[0002]

2. Description of the Related Art It is known that congenital diseases and cancers occur due to abnormalities in the genome of living organisms whose expression is regulated by being methylated. A method for amplifying and analyzing a specific site in the genome has been reported by Nikolai Lisisyn et al. (Science 259, 946-951 (199
3)), and an analysis method based on this was devised and reported by Toshikazu Ushijima et al. (Proceedings of the National Acad).
emy of Sciences of the United States of America Vo
l.94, pp. 2284-2289, March 1997). This method uses a methylation sensitive and insensitive restriction enzyme to cleave a specific part of the genome, and then uses specific primers to
This is a method in which the obtained cleaved fragment is amplified and subjected to electrophoresis or the like for analysis. Also, Minoru Toyota et al.
Methylation sensitive restriction enzyme SmaI and insensitive restriction enzyme Xm
A method for analyzing whether or not a specific site is methylated using aI and an adapter specific to XmaI that produces sticky ends has been reported (Cancer Research 59,
2307-2312 (1999)).

[0003]

However, the conventional detection method can target only a specific methylation site in the genome, and cannot detect methylation of many methylation sites at one time. . An object of the present invention is to provide means and methods for comprehensively analyzing methylation of methylation sites in the genome.

[0004]

The present invention achieves the above object by applying a method for specifically amplifying a methylated site in a genome to a biochip. In order to solve the above-mentioned problems, in the present invention, genomic DNA
Selectively amplify the fragment with methylation sites at both ends,
Biochip, DNA chip, nucleic acid array (hereinafter,
These are collectively called biochips) and devised to be used. This can enable comprehensive analysis of methylation sites.

The biochip for detecting methylation of methylation sites in genomic DNA according to the present invention has a plurality of types of base sequences each containing at least a part of the sequence of a genomic DNA fragment having methylation sites on both ends on a substrate. It is characterized by being fixed at different positions.

[0006] The biochip for detecting methylation of methylation sites in genomic DNA according to the present invention also has both ends cleaved at the recognition sites of a methylation-sensitive restriction enzyme and a methylation-insensitive restriction enzyme having the same recognition site. DNA
A plurality of types of base sequences including at least a part of the fragment sequence are immobilized at different positions on the substrate, respectively.

The method for producing a methylation-detecting biochip used for detecting methylation at a methylation site in genomic DNA according to the present invention is a methylation-insensitive restriction in which a methylation-sensitive restriction enzyme cleaving the same recognition site is present. Digestion of genomic DNA with enzymes and the resulting DNA
The method is characterized by including the steps of amplifying each of the fragments and immobilizing the amplified DNA fragments at different positions on the substrate.

The method for detecting methylated methylation sites in genomic DNA according to the present invention is
In the step of specifically amplifying the nucleotide sequence sandwiched between the two methylated sites, multiple types of nucleotide sequences containing at least part of the sequence of the genomic DNA fragment whose both ends are methylation sites Detecting the amplified base sequence by using biochips fixed at different positions on the substrate.

The step of specifically amplifying the nucleotide sequence sandwiched between the two methylated methylation sites is performed by a methylation-sensitive restriction enzyme and a methylation-insensitive restriction enzyme which cleave the same recognition site, and It is characterized by using an adapter specific to the restriction enzyme. The adapter may be composed of two kinds of oligomers. The two oligomers are preferably unphosphorylated. In addition, two bases are annealed by several bases.

The method for detecting a methylated methylation site in genomic DNA according to the present invention is a method in which a methylation-sensitive restriction enzyme of methylation-sensitive restriction enzyme and methylation-insensitive restriction enzyme that cleaves the same recognition site is used. DNA
And a step of digesting genomic DNA digested with the methylation-sensitive restriction enzyme with the methylation-insensitive restriction enzyme, and a DNA fragment whose both ends are cleavage sites by the methylation-insensitive restriction enzyme And a step of detecting the amplified DNA fragment using a biochip on which a corresponding probe is immobilized on a substrate.

At this time, an enzyme that produces a blunt end is used as a methylation-sensitive restriction enzyme, an enzyme that produces a sticky end is used as a methylation-insensitive restriction enzyme, and both ends are DNA fragments that are cut by the methylation-insensitive restriction enzyme. In the step of specifically amplifying, the PCR can be performed by using an adapter that specifically binds to the sticky end cleaved by the methylation-insensitive restriction enzyme and using the primer portion contained in the adaptor.

[0012] In addition, an enzyme that produces sticky ends was used as both a methylation-sensitive restriction enzyme and a methylation-insensitive restriction enzyme, and genomic DNA was digested with the methylation-sensitive restriction enzyme and then cleaved by the methylation-sensitive restriction enzyme. Including the step of extending the single-stranded portion of the sticky end to a blunt end, the step of specifically amplifying a DNA fragment whose both ends are cleavage sites by a methylation-insensitive restriction enzyme,
PCR can be performed by using an adapter that specifically binds to the sticky end cleaved by a methylation-insensitive restriction enzyme and utilizing the primer portion contained in the adaptor.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. (1) Production of Probe and Biochip A method for producing a biochip probe and biochip will be described with reference to FIG. The biochip probe is a methylation insensitive restriction enzyme XmaI for genomic DNA 100.
It is obtained by digesting (cutting) with. Genomic DNA 10
The XmaI recognition site of 0 has an unmethylated site 101 and a methylated site 102. Digestion with XmaI, a methylation-insensitive restriction enzyme, allows all Xm
The aI recognition site is cleaved. The XmaI recognition site and cleavage pattern are shown in FIG. Genomic DNA produced by this digestion reaction
Fragments 111 to 114 have sticky ends 115.

Next, genomic DNA cleaved by XmaI
The XmaI-specific adapter having the sequence shown in FIG. 3 is ligated to the sticky ends of the fragments 111 to 114. As shown in FIG. 4, the XmaI-specific adapter was prepared by annealing oligonucleotide P1: 5′-AGCACTCTCCAGCCTCTCCGAC-3 ′ and (c) oligonucleotide P2: 5′-CCGGGTCGGTGA-3 ′. In other words, the XmaI-specific adapter is 1) that specifically binds to the XmaI-cleaved fragment, 2) consists of a double-stranded annealed number base, and 3) is phosphorylated so as not to ligate between the adapters. It has not been done.

Next, the method of using the XmaI-specific adapter will be described with reference to FIG. Here, 500 is genomic DNA cleaved by XmaI, 510 is a phosphate group, and 520 is genomic DN.
A represents a connecting portion between the oligonucleotide P1 site of the adapter and 530 represents a connecting portion between the genomic DNA and the oligonucleotide P2 site of the adapter.

XmaI-cleaved genomic DNA 500 and XmaI
The specific adapters 511 and 512 are ligated to react with Xm
Genomic DNA 540 ligated with aI-specific adapter at 72 ℃
Xm bound to genomic DNA fragment after incubation for 5 minutes at
Remove the P2 part of the aI-specific adapter oligonucleotide. Since the oligonucleotide P2 is not phosphorylated, the oligonucleotide P2 site of the adapter cannot be chemically linked to the genomic DNA at the linking portion 530 and it is as short as 12 mer.
Get out of DNA. On the other hand, the genomic DNA and the oligonucleotide P1 linking portion 520 of the adapter are chemically linked by utilizing the phosphate group 510 of the genomic DNA 500 cleaved by XmaI, and are not detached from the genomic DNA by this treatment. . After this, a DNA replication reaction is performed to replicate the DNA complementary to the oligonucleotide P1 part. DNA fragment 550 containing the oligonucleotide P1 part thus created
Corresponds to the part of the genomic DNA sandwiched by the sequences shown in FIG. DN containing this oligonucleotide P1 part
The A fragment 550 can be amplified using the oligonucleotide P1 sequence as a primer.

Returning to FIG. 1, a DNA fragment 121-containing the oligonucleotide P1 part, which was prepared by subjecting the genomic DNA fragments 111-114 cleaved by XmaI to the same treatment.
After being separated, 124 is PCR-amplified by using the oligonucleotide P1 as a primer and used as a probe for a DNA chip. In FIG. 1, the genomic DNA fragment serving as the probe was amplified by PCR, but it is also possible to clone the XmaI fragment. Cloning will be described with reference to FIG.

In this case, genomic DNA 600 is cleaved with XmaI, and the cleaved genomic DNA fragment 610 is a vector having an XmaI restriction enzyme site (for example, pUC18 system when a plasmid is used, and preferably kanamycin resistant pHSG298). , PHSG with chloramphenicol resistance
396, etc., which have antibiotic resistance, etc., and which are easily transformed between a host transformed in the following culture stage and a host not transformed) are prepared, and a recombinant 620 containing an XmaI fragment is prepared. Is introduced into a host (for example, Escherichia coli DH5α) 630 or the like. This host 630
Were cultured in an appropriate selective medium 640, and single colonies 641 that had grown were picked up.
This means that a host having a vector into which was incorporated could be selected. At this time, colony hybridization can be performed to select a host having a specific DNA.

A clone of the Xma I fragment can be obtained by the above operation, and a sample Xma I fragment library can be prepared if necessary. Further, by using the primer 625 of the vector for PCR, this DNA can be easily and repeatedly used. It is also possible to determine the base sequence of a part of the DNA fragment obtained by such a method using a sequencer or the like, synthesize the sequence, and use this as a probe.

A biochip is prepared by dissolving the DNA obtained by the above-mentioned method or a synthesized part of the sequence of the DNA in an appropriate solvent and fixing it on a substrate as a probe. As the solvent, for example, sterile distilled water, a buffer such as TE or SSC, a polymer such as cellulose, an organic solvent such as DMSO, or a mixture thereof can be used.

In producing a biochip, as shown in FIG. 1, the DNA solution is spotted on a membrane or a slide glass, which will be a substrate 130 of the biochip, with a thin pin 131 for the biochip, for example. Board 130
Is coated with polylysine or silane to facilitate DNA binding. A commercially available biochip manufacturing apparatus can be used for manufacturing the biochip.
The spotted probe DNA is fixed on the substrate 130 by an appropriate method and then used for hybridization with the target.

(2) Preparation of Sample (Target) Next, a method for preparing a genomic sample for which a methylation site is to be analyzed using the biochip produced as described above will be described. Here, SmaI and XmaI are used as a methylation sensitive restriction enzyme and an insensitive restriction enzyme that cut the same recognition site, and a genomic DNA is used by using an adapter specific for XmaI.
The method for amplifying the portion sandwiched between the SmaI and XmaI recognition sites that are methylated in is described.

FIG. 7 shows SmaI and X contained in genomic DNA.
It is explanatory drawing of the method of amplifying the part flanked by the methylation methylation site among maI recognition sites.
Unmethylated site 701 which is cleaved by SmaI and XmaI, and Xm
Genomic DNA 7 containing a methylation site 702 cleaved by aI
00 is first digested with the methylation sensitive restriction enzyme SmaI. The SmaI recognition site and cleavage pattern are as shown in FIG. 8, and the SmaI-digested genomic DNA fragment 710 formed by this digestion reaction has blunt ends 711. Next, the SmaI-digested genomic DNA 710 digested with SmaI is further digested with a methylation-insensitive restriction enzyme XmaI. XmaI digested genomic DNA fragment 720 formed by this digestion reaction has sticky ends
It is 721.

Next, the XmaI-specific adapter described in FIG. 3 is ligated to the sticky end 721 of the XmaI-cleaved genomic DNA fragment. At this time, this adapter does not ligate to the blunt end 711 of the genomic DNA fragment cleaved by SmaI. After this, as explained in FIGS. 1 and 5, genomic DNA with XmaI-specific adapters ligated at both ends.
Is incubated at 72 ° C. for 5 minutes, the oligonucleotide P2 portion of the XmaI-specific adapter bound to the genomic DNA fragment is removed, and the portion is extended. DNA fragments 731 and 732 containing the P1 portion of the oligonucleotide thus created
2 corresponds to a part of genomic DNA 700 which is sandwiched at both ends by the sequence shown in FIG. 2 and both ends thereof are methylated. The DNA fragments 731 and 732 containing the oligonucleotide P1 part can be amplified by using the oligonucleotide P1 sequence as a primer, and when amplified, a fluorescent substance, for example, a biochip target by labeling the DNA with Cy3 or Cy5. To be done. Examples of the labeling method include a method such as random priming.

FIG. 9 is an explanatory diagram in the case where two kinds of enzymes used for preparing a sample are those which generate sticky ends. First, genomic DNA 900 is first digested with a methylation sensitive enzyme. The methylation-sensitive enzyme-digested genomic DNA 910 thus obtained has sticky ends 911. Next, the single-stranded portion of the generated sticky end 911 is extended with a DNA polymerase such as Klenow fragment to make a blunt end 912.

Then, when digested with the other methylation-insensitive enzyme, the resulting methylation-insensitive enzyme-digested genomic DNA 920 has sticky ends 921. Therefore, a specific adapter was ligated to the latter methylation-insensitive enzyme, the same reaction as described in FIGS. 1 and 5 was performed, and the primer portion contained in the specific adapter was used to generate PC.
When R is performed, DNA that is methylated at both ends as a PCR product
Can be obtained.

The detection of the methylation site in the genomic DNA of newborn mice and ES cells carried out by the method of the present invention will be described below. FIG. 10 is a schematic explanatory diagram showing a procedure for detecting a methylation site.

The biochip 1000 was prepared by using polylysine-coated glass as a substrate and spotting probe DNA 10 samples × 4 times. Biochip 10
Each DNA spotted at 00 is the genomic DNA of newborn mouse
It is an XmaI fragmented and amplified product, that is, an XmaI fragment of genomic DNA containing the above-mentioned methylation site, and can be examined for methylation and non-methylation.

Mouse neonatal genomic DNA 1010 and ES
Treatment 1100 of digesting cell genomic DNA 1020 separately with methylation-sensitive restriction enzyme SmaI, treatment 1200 of digesting with methylation-insensitive restriction enzyme XmaI, Xm
A treatment 1300 for ligating an aI-specific adapter, a PCR for labeling and amplification, and a labeling reaction 1400 were performed. Using the random primer method described with reference to FIG. 7, the target mouse genomic DNA 1010 was Cy3, ES.
Cell genomic DNA 1020 was labeled with Cy5. After hybridization 1500, the slide glass was washed to remove the background and fluorescence was detected.

In the upper part of FIG. 11, the mouse genomic DNA DN is shown.
The fluorescence intensity derived from A (Cy3) and the fluorescence intensity derived from genomic DNA of ES cells (Cy5) are shown in the middle row. Probe on biochip
The arrangement of DNA is shown in the lower part of FIG. Spots with strong fluorescence appear white, and as the fluorescence weakens, they are gradually displayed darker.

The fluorescence intensity is digitized and graphed as shown in FIG. 12. The horizontal axis of the graph shows the probe DNA numbers 1 to 10 and the left side of the vertical axis is the ratio of the fluorescence intensity of Cy5 to the fluorescence intensity of Cy3 = Cy5 / Cy3 is shown, and the distance from the origin of each spot on the scatter diagram on the right side of the vertical axis = [(Cy3 fluorescence intensity) ² +
(Cy5 fluorescence intensity) ² ] ^1/2 is shown. Of the 10 samples x 4 spots, the distance from the origin is shown for one set and Cy5 / Cy3 for all 4 sets.

From FIG. 12, it can be seen that, for DNA No. 7, the fluorescence intensity of Cy3 is about twice higher than that of Cy5. This makes this DNA a newborn mouse genomic DNA.
Was found to be methylated about twice as high as that of ES cell genomic DNA. Also, this was similar to the results obtained by the conventional method.

[0033]

Industrial Applicability According to the present invention, it becomes possible to comprehensively analyze methylation sites in the genome.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a biochip probe and a method for producing a biochip.

FIG. 2 is a view showing a recognition sequence of XmaI and a cleavage pattern.

FIG. 3 shows the sequence of the XmaI-specific adapter.

FIG. 4 is a view showing a method for preparing an XmaI-specific adapter.

FIG. 5 is a view showing a method of using an XmaI-specific adapter.

FIG. 6 is an explanatory view of a method for producing a probe for biochip and its cloning.

FIG. 7 is an explanatory view of a method for amplifying a portion sandwiched between sequences that are methylated in genomic DNA.

FIG. 8 shows SmaI recognition sequences and cleavage patterns.

FIG. 9 is a diagram showing a method for preparing a biochip target using a restriction enzyme that produces cohesive ends.

FIG. 10 is a schematic explanatory view showing a procedure for detecting a methylation site.

FIG. 11 is a diagram showing a hybridization fluorescence detection image and the arrangement of probe DNAs.

FIG. 12 is a graph in which fluorescence intensity is digitized and graphed.

[Explanation of symbols]

100 ... Genomic DNA, 111 ... Unmethylated site, 112
... Methylation site, 111-114 ... Genomic DNA fragment cut by XmaI, 115 ... Sticky end, 130 ... Substrate, 131 ... Pin, 500 ... Genome D cut by XmaI
NA, 510 ... Phosphate group, 511, 512 ... XmaI specific adapter, 550 ... DNA containing oligonucleotide P1 part
Fragment, 600 ... Genomic DNA, 610 ... Genomic DNA fragment cleaved with XmaI, 620 ... Recombinant containing XmaI fragment, 6
25 ... Vector primer, 630 ... Host, 640 ...
Selection medium, 641 ... Single colony, 700 ... Genome
DNA, 701 ... Unmethylated site, 702 ... Methylated site, 711 ... Blunt end, 721 ... Sticky end, 900 ... Genomic DNA, 910 ... Methylation sensitive enzyme digested genomic DNA,
911 ... Sticky end, 912 ... Blunt end, 920 ... Methylation insensitive enzyme digested genomic DNA, 920 ... Sticky end, 9
31, 932 ... Adapter specific to methylation insensitive enzyme, 1000 ... Biochip, 1100 ... Digestion with methylation-sensitive restriction enzyme SmaI, 1200 ... Digestion with methylation-insensitive restriction enzyme XmaI, 1300 ... XmaI-specific adapter ligation Treatment, 1400 ... PCR / labeling reaction, 15
00 ... Hybridization

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/566 C12N 15/00 ZNAA 37/00 102 F (72) Inventor Deho Hatada Maebashi, Gunma Prefecture Shimoko 2-51-25 Demachi Sun Palace Maebashi 801 (72) Inventor Atsumi Tsujimoto 134 Kobe-cho, Hodogaya-ku, Yokohama-shi, Kanagawa Yokohama Business Park East Tower 14F DNAC Inc. (72) Inventor Azusa Kato Kanagawa 14th floor, Yokohama Business Park East Tower, 14th floor, Kobe-cho, Hodogaya-ku, Yokohama-shi, Japan (72) Inventor, NAIPIC Laboratories, Inc. (72) Iko Yurino 134th floor, Kobe-cho, Hodogaya-ku, Yokohama-shi, Kanagawa Yokohama Business Park East Tower, 14th floor Company Nichichi Institute within the F-term (reference) 4B024 AA11 CA01 CA09 HA09 HA12 HA20 4B029 AA07 AA23 BB20 CC03 CC08 FA15 4B063 QA01 QA13 QQ42 QR14 QR32 QR56 QR62 QR84 QS25 QS34

Claims (11)

[Claims] 1. A methylation site in a genomic DNA, characterized in that a plurality of types of nucleotide sequences containing at least a part of the sequence of a genomic DNA fragment having methylation sites at both ends are immobilized at different positions on a substrate, respectively. A biochip for the detection of methylation. 2. A substrate having a plurality of types of nucleotide sequences each containing at least a part of a sequence of a DNA fragment whose both ends are cut at the recognition sites of a methylation-sensitive restriction enzyme and a methylation-insensitive restriction enzyme having the same recognition site. A biochip for detecting methylation of methylation sites in genomic DNA, characterized by being immobilized at different positions above. 3. A method for producing a biochip for detecting methylation, which is used for detecting methylation at a methylation site in genomic DNA, comprising a methylation insensitive enzyme having a methylation sensitive restriction enzyme cleaving the same recognition site. A method for detecting methylation, comprising the steps of digesting genomic DNA with a restriction enzyme, amplifying each of the obtained DNA fragments, and immobilizing the amplified DNA fragments at different positions on the substrate. Biochip manufacturing method. 4. A method for detecting a methylated methylation site in genomic DNA, which comprises specifically amplifying a nucleotide sequence flanked by two methylated sites in genomic DNA; A plurality of kinds of base sequences containing at least a part of the sequence of the genomic DNA fragment which is a methylation site are detected at different positions on the substrate, respectively, using the biochip to detect the amplified base sequence, A method comprising. 5. The method according to claim 4, wherein the step of specifically amplifying the nucleotide sequence sandwiched between the two methylated methylation sites is a methylation-sensitive restriction enzyme that cleaves the same recognition site. And a methylation-insensitive restriction enzyme, and an adapter specific to either restriction enzyme. 6. The method according to claim 5, wherein the adapter comprises two oligomers. 7. The method of claim 6, wherein the two oligomers are unphosphorylated. 8. The method according to claim 6, wherein the two kinds of oligomers are annealed by several bases. 9. A method for detecting a methylated methylation site in genomic DNA, which comprises using a methylation-sensitive restriction enzyme of a methylation-sensitive restriction enzyme and a methylation-insensitive restriction enzyme that cleaves the same recognition site. And a step of digesting genomic DNA digested with the methylation-sensitive restriction enzyme with the methylation-insensitive restriction enzyme, and a DNA fragment whose both ends are cleavage sites by the methylation-insensitive restriction enzyme And a step of detecting the amplified DNA fragment using a biochip on which a corresponding probe is immobilized on a substrate. 10. The method according to claim 9, wherein the methylation-sensitive restriction enzyme is an enzyme that produces blunt ends.
The methylation-insensitive restriction enzyme is an enzyme that produces cohesive ends, and in the step of specifically amplifying a DNA fragment whose both ends are cleavage sites by the methylation-insensitive restriction enzyme, A method comprising performing PCR using an adapter that specifically binds to the cleaved cohesive end and utilizing a primer portion contained in the adaptor. 11. The method according to claim 9, wherein both the methylation-sensitive restriction enzyme and the methylation-insensitive restriction enzyme are enzymes that generate cohesive ends, and genomic DNA was digested with the methylation-sensitive restriction enzyme. After that, the step of extending the single-stranded portion of the sticky end cleaved by the methylation-sensitive restriction enzyme to make a blunt end, wherein the both ends are specific to the DNA fragment that is a cleavage site by the methylation-insensitive restriction enzyme In the step of amplifying to, a method characterized in that an adapter that specifically binds to the sticky end cleaved by the methylation-insensitive restriction enzyme is used, and PCR is carried out using the primer portion contained in the adaptor.