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WO2017104675A1 - Procédé de préparation d'échantillon pour analyse de méthylation d'adn - Google Patents

Procédé de préparation d'échantillon pour analyse de méthylation d'adn Download PDF

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WO2017104675A1
WO2017104675A1 PCT/JP2016/087100 JP2016087100W WO2017104675A1 WO 2017104675 A1 WO2017104675 A1 WO 2017104675A1 JP 2016087100 W JP2016087100 W JP 2016087100W WO 2017104675 A1 WO2017104675 A1 WO 2017104675A1
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dna
sample
pcr
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航 藤渕
順子 山根
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Kyoto University NUC
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to an improved DNA methylation analysis method. Specifically, the present invention relates to a method for increasing the yield of a DNA sample capable of PCR amplification after bisulfite treatment in DNA methylation analysis using bisulfite.
  • DNA methylation is one of epigenetic mechanisms and is deeply involved in embryogenesis, cell differentiation, phenotypic differences, various diseases, and the like.
  • various methods for DNA methylation analysis have been developed and used.
  • DNA methylation analysis has also been used to select iPS cells that are suitable for the purpose.
  • a method using bisulfite treatment is frequently used as a DNA methylation analysis method for a specific genomic region.
  • Bisulfite converts unmethylated cytosine in DNA to uracil, but methylated cytosine is not converted to other bases. Utilizing this fact, the DNA methylation state can be known by performing sequence analysis after PCR amplification of the bisulfite-treated DNA.
  • the RRBS method is a method capable of examining DNA methylation at a single base level, can reduce the sequence amount, and can obtain the same coverage as the whole genome analysis method.
  • attempts have been made to perform methylation analysis of single-cell DNA using the BBRS method (see Non-Patent Documents 1 and 2).
  • the bisulfite treatment is performed under severe conditions, so the DNA sample is fragmented and a sufficient amount for PCR amplification in the next step is obtained. There is a problem that DNA is not supplied.
  • the problem to be solved by the present invention was to develop an effective and simple method for increasing the yield of a DNA sample capable of PCR amplification after bisulfite treatment.
  • the present inventors have intensively studied to solve the above-mentioned problems, and can perform PCR amplification after bisulfite treatment by performing rescue including a step of treating a DNA sample after bisulfite treatment with single-stranded DNA ligase.
  • the inventors have found that the yield of a simple DNA sample can be increased, and have completed the present invention.
  • the present invention relates to the following.
  • (1) A method for DNA methylation analysis using bisulfite treatment, wherein the DNA sample after bisulfite treatment is treated with a single-stranded DNA ligase, and thereby a DNA sample capable of PCR amplification after bisulfite treatment
  • (2) The method according to (1), wherein the DNA methylation analysis using bisulfite treatment is based on the RRBS (Reduced Representation Bisulfite Sequencing) method.
  • the DNA sample after the bisulfite treatment is subjected to the following steps: (A) The method according to (1) or (2), which is subjected to a treatment with DNA 5′-, 3′-phosphatase or DNA 3′-phosphatase, and (b) a treatment with DNA 5′-kinase. (4) The method according to any one of (1) to (3), wherein the DNA sample after the bisulfite treatment is immobilized on a carrier. (5) The method according to any one of (1) to (4), wherein step (b) is performed in the presence of ATP. (6) A kit for carrying out the method according to any one of (1) to (5), comprising a single-stranded DNA ligase. (7) The kit according to (6), further comprising DNA 5′-, 3′-phosphatase or DNA 3′-phosphatase and DNA 5 ′ kinase.
  • the yield of a PCR-amplifiable DNA sample after bisulfite treatment can be increased by a simple method using a single-stranded DNA ligase, and an amount sufficient for the subsequent PCR and sequencing steps.
  • a DNA sample can be supplied. Therefore, if this invention is used, methylation analysis of a trace amount DNA sample, such as DNA derived from a single cell, will be attained.
  • FIG. 1 is a graph showing the effect of treatment with single-stranded DNA ligase.
  • the horizontal axis represents the sample name, and the vertical axis represents the DNA amount (ng / ⁇ l).
  • Sample I was not treated with bisulfite treatment and treatment with single-stranded DNA ligase
  • Sample IV was treated with bisulfite treatment but was not treated with single-stranded DNA ligase
  • Sample V was bisulfite
  • the amount of PCR amplification product of the sample subjected to the fight treatment and the treatment with single-stranded DNA ligase is shown.
  • FIG. 2 is a photograph showing the results of electrophoresis using a 2.0% E-gel of the PCR amplification product described in FIG.
  • FIG. 3 is a graph showing DNA yield results when a human iPS cell line is used as a sample.
  • BS (+) Res (-) is bisulfite treatment only
  • BS (+) Res (+) is subjected to bisulfite treatment. This is the case.
  • FIG. 4 is a graph showing the results of DNA yield when a human ES cell line 12 single cell is used as a sample.
  • FIG. 5 shows the result of analyzing the sequence obtained from the DNA obtained from the human ES cell line by the next generation sequence.
  • the left figure shows the case where the method of the present invention is not applied, and the right figure shows the case where the method of the present invention is applied.
  • FIG. 6 shows a Lorentz curve when DNA obtained from a human ES cell line was sequenced and analyzed by the next generation sequence. The left figure shows the case where the method of the present invention is not applied, and the right figure shows the case where the method of the present invention is applied.
  • FIG. 5 shows the result of analyzing the sequence obtained from the DNA obtained from the human ES cell line by the next generation sequence.
  • the left figure shows the case where the method of the present invention is not applied, and the right figure shows the case where the method of the present invention is applied.
  • FIG. 7 is a graph showing the yield results when human genomic DNA is used as a sample.
  • BS (+) Res (-) is bisulfite treatment only
  • BS (+) Res (+) is subjected to bisulfite treatment.
  • BS (+) Res (ATP) is the case where ATP is added during the kinase treatment.
  • the present invention is a method for DNA methylation analysis using bisulfite treatment, wherein a DNA sample after bisulfite treatment is treated with a single-stranded DNA ligase, thereby
  • the present invention relates to a method characterized by increasing the yield of a PCR-amplifiable DNA sample.
  • DNA methylation analysis using bisulfite treatment is widely used.
  • Bisulfite converts unmethylated cytosine in DNA to uracil, but methylated cytosine is not converted to other bases. Utilizing this fact, the DNA methylation state can be known by performing sequence analysis after PCR amplification of the bisulfite-treated DNA.
  • the bisulfite itself is a highly reactive substance, and the processing conditions are also high. Therefore, when the bisulfite treatment is performed, the DNA sample is fragmented and a sufficient amount of DNA is supplied for the next PCR amplification. There is a problem that it is not.
  • the present invention performs rescue by subjecting the DNA sample after the bisulfite treatment to a single-stranded DNA ligase treatment, and a sufficient amount for PCR amplification as the next step. It supplies DNA.
  • rescue has not been performed in DNA methylation analysis using conventional bisulfite treatment.
  • DNA methylation analysis methods using bisulfite treatment examples include the RRBS method and the whole genome bisulfite sequencing (WGBS) method.
  • the present invention can be carried out in any DNA methylation analysis method using bisulfite, but is preferably carried out in the RRBS method. In the following, the invention will be described in relation to the RRBS method.
  • the RRBS method includes the following steps (see H. Guo et al. Nature Protocols, 10 (5), 645-659 (2015), etc.): Digestion of DNA samples with restriction enzymes End repair and dA tailing Adapter ligation Bisulfite treatment PCR amplification Sequencing
  • a DNA sample is rescued by ligating a fragmented DNA sample using a single-stranded DNA ligase between bisulfite treatment and PCR amplification.
  • the single-stranded DNA ligase used here may be any enzyme as long as it has an activity of ligating single-stranded DNA, and is also commercially available.
  • Examples of the single-stranded DNA ligase used in the present invention include, but are not limited to, Epicentre's CircLigase TM ssDNA Ligase, Wako Pure Chemical Industries, Ltd., Single Strand DNA Ligase, thermostable, recombinant, Solution, and the like.
  • Preferred reaction conditions for single-stranded DNA ligase vary depending on the type of enzyme, but they are known to those skilled in the art or can be easily determined by those skilled in the art.
  • single-stranded DNA ligase catalyzes a reaction of binding a hydroxyl group at the 3 'end of a polynucleotide and a phosphate group at the 5' end. Therefore, the 3 ′ end of the fragmented DNA sample after the bisulfite treatment has a hydroxyl group and a phosphate group is added to the 5 ′ end, so that the efficiency of the ligation reaction by single-stranded DNA ligase and the ligated DNA This leads to an increase in yield.
  • treatment with DNA 5'-, 3'-phosphatase or DNA 3'-phosphatase and treatment with DNA 5'-kinase are preferably performed before the ligation reaction with single-stranded DNA ligase.
  • the DNA 5′-, 3′-phosphatase used in the present invention is any one as long as it has an activity of hydrolyzing the phosphate groups at the 5 ′ end and 3 ′ end of DNA to generate hydroxyl groups.
  • ThermoFisher SCIENTIFIC product number EF0654 may be used, but the present invention is not limited thereto.
  • Preferred reaction conditions for DNA 5'-, 3'-phosphatase vary depending on the type of enzyme, but these are known to those skilled in the art or can be easily determined by those skilled in the art.
  • the DNA 3'-phosphatase used in the present invention may be any DNA as long as it has an activity of hydrolyzing a phosphate group at the 3 'end of DNA to generate a hydroxyl group.
  • Preferred reaction conditions for DNA 3'-phosphatase vary depending on the type of enzyme, but these are known to those skilled in the art or can be easily determined by those skilled in the art.
  • Polynucleotide kinase 3'-phosphatase (PNKP) may also be used.
  • PNKP is an enzyme having 3 'phosphatase activity and 5'-kinase activity.
  • PNKPs are commercially available from QIAGEN.
  • the 5′-kinase used in the present invention may be any as long as it has an activity of adding a phosphate group to the 5 ′ end of DNA, such as T4 polynucleotide kinase manufactured by Promega.
  • Preferred reaction conditions for DNA5'-kinase vary depending on the type of enzyme, but these are known to those skilled in the art or can be easily determined by those skilled in the art.
  • the yield of the DNA sample can be further increased by conducting the kinase reaction in the presence of ATP.
  • the amount of ATP is not particularly limited and can be appropriately determined by those skilled in the art. Examples of the amount of ATP include, but are not limited to, a final concentration of about 1 mM to several mM in the kinase reaction system.
  • DNA 5′-, 3′-phosphatase When DNA 5′-, 3′-phosphatase is used, the DNA 5′-, 3′-phosphatase is allowed to act before allowing DNA 5′-kinase to act, and the 5 ′ end of the fragmented DNA sample generated by bisulfite treatment is used. And the phosphate group at both 3 'ends is hydrolyzed to give a hydroxyl group at each end. Next, DNA 5'-kinase is allowed to act to add a phosphate group to the 5 'end of the fragmented DNA sample. Thereafter, a single-stranded DNA ligase is allowed to act, and the fragmented DNA samples are ligated and rescued.
  • DNA 3 ′ phosphatase When DNA 3 ′ phosphatase is used instead of DNA 5′-, 3′-phosphatase, (i) treatment with DNA 3 ′ phosphatase and treatment with DNA 5′-kinase may be performed simultaneously.
  • the treatment (i) may be performed using a NEB T4 polynucleotide kinase.
  • DNA 3′-phosphatase has negligible activity to hydrolyze the phosphate group at the 5 ′ end of DNA, or such It is necessary to have no activity.
  • the DNA sample In order to efficiently perform the ligation reaction with single-stranded DNA ligase and increase the yield of the rescued DNA sample, it is preferable to bind the DNA sample to an immobilization carrier. By collecting the carrier on which the DNA sample is immobilized, the DNA sample is concentrated and purified, the rescue efficiency is increased, and more DNA samples can be recovered.
  • the DNA sample may be immobilized either before or after the bisulfite treatment, but preferably after the bisulfite treatment. The reason is that the immobilization support may interfere with the reaction during the reaction during the bisulfite treatment, and the purification after the bisulfite treatment uses a column, and thus the immobilization support cannot pass through the column. .
  • a substance or functional group capable of binding to the carrier can be bound or introduced into the adapter.
  • An adapter to which such a substance or functional group has been bound or introduced in advance may be linked to the DNA sample, or such substance or functional group may be bound or introduced after the adapter has been linked to the DNA sample.
  • the adapter design is known to those skilled in the art, and can be performed according to the conditions such as the base sequence of the primers used in PCR amplification and the sequencer. You may use the adapter contained as a component of the commercial kit for methylation analysis.
  • Substances to be bound to the adapter for immobilizing the DNA sample and functional groups to be introduced to the adapter are known to those skilled in the art, and their binding method, introduction method, and immobilization method to the carrier are also known to those skilled in the art. is there.
  • biotin can be added to the end of the adapter, avidin bound to the carrier, and immobilization can be performed using a biotin-avidin bond.
  • magnetic beads bound with avidin are suitable as the carrier.
  • the present invention provides a method for increasing the yield of a DNA sample capable of PCR amplification after bisulfite treatment in DNA methylation analysis using bisulfite, wherein the DNA sample after bisulfite treatment is single-stranded.
  • the present invention relates to a kit for carrying out a method characterized by treatment with DNA ligase. Therefore, the kit of the present invention contains a single-stranded DNA ligase.
  • the kit of the present invention may further contain DNA 5'-, 3'-phosphatase or DNA 3'-phosphatase, and DNA 5'-kinase.
  • Single-stranded DNA ligase, DNA5'-, 3'-phosphatase, DNA3'-phosphatase, and DNA5'-kinase are as described above.
  • the rescue reaction of the present invention was performed after bisulfite treatment, and the increase in the yield of the recovered DNA sample was examined.
  • the rescue reaction included four steps 5-1), 5-2), 5-3) and 5-4).
  • the beads were washed twice with 1 ⁇ BWT buffer (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.02% Tween-20), and then EBT buffer (10 mM Tris-HCl (pH 8)). 5), 0.02% Tween-20) and washed once.
  • 1 ⁇ BWT buffer 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.02% Tween-20
  • EBT buffer 10 mM Tris-HCl (pH 8)
  • phosphatase reaction A phosphatase mixture was prepared using 1x buffer and nuclease-free water. The beads of 5-1) were captured and the supernatant was removed, and then suspended in a phosphatase mixture and transferred to a PCR tube. 1 ⁇ L of FastAP Thermosensitive Alkaline Phosphatase (ThermoFisher SCIENTIFIC product number EF0654) was added, reacted at 37 ° C. for 10 minutes, and enzyme-inactivated at 75 ° C. for 5 minutes. After the reaction, it was transferred to a LoBind tube to capture the beads, and washed twice with EBT buffer.
  • FastAP Thermosensitive Alkaline Phosphatase Thermosensitive Alkaline Phosphatase
  • kinase reaction A kinase mixture was prepared using 1x buffer and nuclease-free water. The beads of 5-2) were captured and the supernatant was removed, and then suspended in a kinase mixture and transferred to a PCR tube. 1 ⁇ L of T4 polynucleotide kinase (TaKaRa product number 2021A) was added, reacted at 37 ° C. for 30 minutes, and enzyme-inactivated at 70 ° C. for 5 minutes. After the reaction, it was transferred to a LoBind tube to capture the beads, and washed twice with EBT buffer.
  • T4 polynucleotide kinase T4 polynucleotide kinase
  • the PCR reaction was performed as follows. 95 ° C. for 2 minutes ⁇ 95 ° C. for 20 seconds for 22 cycles, 60 ° C. for 30 seconds, 72 ° C. for 1 minute ⁇ 72 ° C. for 5 minutes ⁇ 4 ° C.
  • Dynabeads® M-280 streptavidin beads were captured, the supernatant was collected, and the PCR product was purified twice with 1: 1 AMPure beads (see Guo et al., Supra).
  • Second round PCR enrichment A PCR mixture was created as follows. 1 ⁇ Phusion HF PCR master mix with HF buffer (see Guo et al., Supra), 500 nM primer (sequence above), and sample. Thereafter, the PCR reaction was performed as follows. 98 ° C. for 2 minutes ⁇ 98 ° C. for 10 seconds for 22 cycles, 60 ° C. for 30 seconds, 72 ° C. for 1 minute ⁇ 72 ° C. for 5 minutes ⁇ 4 ° C. After completion of the PCR reaction, the PCR product was purified once with 1: 1 AMPure beads, eluted with 25 ⁇ L of nuclease-free water and collected.
  • the PCR product measurement results are shown in Table 2 and FIG.
  • the results of electrophoresis with 2.0% E-gel are shown in FIG. 2 (the center lane is sample I, the second lane from the right is sample IV, and the rightmost lane is sample V). According to the comparison between Sample IV and Sample V, a yield increase of about 2.3 times was recognized by the rescue reaction of the present invention.
  • 201B7 cell line which is a human iPS cell line, was used as a sample. After culturing the 201B7 cell line on feeder cells, the cells were collected, and the cell suspension stained with anti-SSEA-4 antibodies (BD Pharmingen) and PI (BD Pharmingen) was used in single cell mode using FACS Aria II (BD Biosciences). Then, anti-SSEA-4 antibody positive and PI negative cells were sorted in a 96-well PCR plate by 300 cells.
  • the experimental procedure included the following seven steps: 1) digestion with MspI, 2) end repair / dA tailing reaction, 3) adapter ligation, 4) bisulfite treatment, 5) rescue reaction, 6) first round. PCR enrichment, and 7) Second round PCR enrichment. In this additional data, 8) Quantitative PCR was carried out using human-specific gene primers, using the gel sample, the purified sample as a sequence, and the purified sample as a template.
  • a Y-shaped adapter containing a biotinylated barcode on both sides of the following sequence was used as an adapter.
  • a Y-shaped adapter was prepared by annealing a universal adapter with a biotinylated 5 ′ end and an index adapter with a biotinylated 3 ′ end.
  • the sequence of the universal adapter is as follows.
  • the final elution used 25 ⁇ L of elution buffer preheated at 50 ° C.
  • the amount was measured and divided into two equal parts (newly sample II (BS (+) / rescue ( ⁇ ) in Table 3, BS (+) Res ( ⁇ ) in FIG. 3)), sample III (BS (+) in Table 3) / Rescue (+), named BS (+) Res (+)) in FIG.
  • the rescue reaction included four steps 5-1), 5-2), 5-3) and 5-4).
  • the beads were washed twice with 1 ⁇ BWT buffer (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.02% Tween-20), and then EBT buffer (10 mM Tris-HCl (pH 8)). 5), 0.02% Tween-20) and washed once.
  • 1 ⁇ BWT buffer 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.02% Tween-20
  • EBT buffer 10 mM Tris-HCl (pH 8)
  • phosphatase reaction A phosphatase mixture was prepared using 1x buffer and nuclease-free water. The beads of 5-1) were captured and the supernatant was removed, and then suspended in a phosphatase mixture and transferred to a PCR tube. 1 ⁇ L of FastAP Thermosensitive Alkaline Phosphatase (ThermoFisher SCIENTIFIC product number EF0654) was added, reacted at 37 ° C. for 10 minutes, and enzyme-inactivated at 75 ° C. for 5 minutes. After the reaction, it was transferred to a LoBind tube to capture the beads, and washed twice with EBT buffer.
  • FastAP Thermosensitive Alkaline Phosphatase Thermosensitive Alkaline Phosphatase
  • kinase reaction A kinase mixture was prepared using 1x buffer and nuclease-free water. The beads of 5-2) were captured and the supernatant was removed, and then suspended in a kinase mixture and transferred to a PCR tube. 1 ⁇ L of T4 polynucleotide kinase (TaKaRa product number 2021A) was added, reacted at 37 ° C. for 30 minutes, and enzyme-inactivated at 70 ° C. for 5 minutes. After the reaction, it was transferred to a LoBind tube to capture the beads, and washed twice with EBT buffer.
  • T4 polynucleotide kinase T4 polynucleotide kinase
  • Second round PCR enrichment A PCR mixture was created as follows. 1 ⁇ Phusion HF PCR master mix with HF buffer (see Guo et al. Literature), 500 nM primer (see Guo et al. Literature), and sample. Thereafter, the PCR reaction was performed as follows. Set at 98 ° C. for 2 minutes ⁇ 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute 22 cycles ⁇ 72 ° C. for 5 minutes ⁇ 4 ° C. After completion of the PCR reaction, the PCR product was purified once with 1: 1 AMPure beads, eluted with 25 uL of nuclease-free water and collected.
  • Quantitative PCR was performed with human-specific gene primers using the gel sample (250-700 bp) and purified sample as a template.
  • the primer sequences used are as follows. 5'-TAGCAATAATCCCCATCCTCCATATAT-3 '(SEQ ID NO: 6) 5'-ACTTGTCCAATGATGGTAAAAGG-3 '(SEQ ID NO: 7)
  • Adapter ligation 12 types of adapters with biotinylated barcodes were added to each well.
  • 30 Weiss units of T4 DNA ligase, 1 ⁇ Tango buffer, 1 mM ATP, 12 types of adapters, nuclease-free water were added, reacted at 16 ° C. for 30 minutes, and subsequently reacted at 4 ° C. overnight, then at 65 ° C.
  • the enzyme was inactivated for 20 minutes. The exact amount of sample was measured and adjusted with nuclease-free water to 25 ⁇ L.
  • a Y-shaped adapter containing a biotinylated barcode on both sides of the following sequence was used as an adapter.
  • a Y-shaped adapter was prepared by annealing a universal adapter with a biotinylated 5 ′ end and an index adapter with a biotinylated 3 ′ end.
  • the sequence of the universal adapter is as follows.
  • sample I (again, BS (+) / rescue ( ⁇ ) in Table 4, FIG. 4 BS (+) Res ( ⁇ )), sample II (named BS (+) / rescue (+) in Table 4, BS (+) Res (+)) in FIG. 4).
  • the rescue reaction included four steps 5-1), 5-2), 5-3) and 5-4).
  • the beads were washed twice with 1 ⁇ BWT buffer (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.02% Tween-20), and then EBT buffer (10 mM Tris-HCl (pH 8)). 5), 0.02% Tween-20) and washed once.
  • 1 ⁇ BWT buffer 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.02% Tween-20
  • EBT buffer 10 mM Tris-HCl (pH 8)
  • phosphatase reaction A phosphatase mixture was prepared using 1x buffer and nuclease-free water. The beads of 5-1) were captured and the supernatant was removed, and then suspended in a phosphatase mixture and transferred to a PCR tube. 1 ⁇ L of FastAP Thermosensitive Alkaline Phosphatase (ThermoFisher SCIENTIFIC product number EF0654) was added, reacted at 37 ° C. for 10 minutes, and enzyme-inactivated at 75 ° C. for 5 minutes. After the reaction, it was transferred to a LoBind tube to capture the beads, and washed twice with EBT buffer.
  • FastAP Thermosensitive Alkaline Phosphatase Thermosensitive Alkaline Phosphatase
  • kinase reaction A kinase mixture was prepared using 1x buffer and nuclease-free water. The beads of 5-2) were captured and the supernatant was removed, and then suspended in a kinase mixture and transferred to a PCR tube. 1 ⁇ L of T4 polynucleotide kinase (TaKaRa product number 2021A) was added, reacted at 37 ° C. for 30 minutes, and enzyme-inactivated at 70 ° C. for 5 minutes. After the reaction, it was transferred to a LoBind tube to capture the beads, and washed twice with EBT buffer.
  • T4 polynucleotide kinase T4 polynucleotide kinase
  • Second round PCR enrichment A PCR mixture was created as follows. 1 ⁇ Phusion HF PCR master mix with HF buffer (see Guo et al. Literature), 500 nM primer (see Guo et al. Literature), and sample. Thereafter, the PCR reaction was performed as follows. Set at 98 ° C. for 2 minutes ⁇ 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute 26 cycles ⁇ 72 ° C. for 5 minutes ⁇ 4 ° C. After completion of the PCR reaction, the PCR product was purified once with 1: 1 AMPure beads, eluted with 25 uL of nuclease-free water and collected.
  • Quantitative PCR was performed with human-specific gene primers using the gel sample (250-700 bp) and purified sample as a template.
  • the primer sequences used are as follows. 5'-TAGCAATAATCCCCATCCTCCATATAT-3 '(SEQ ID NO: 6) 5'-ACTTGTCCAATGATGGTAAAAGG-3 '(SEQ ID NO: 7)
  • the obtained gene sequence was analyzed using a next-generation sequencer (Illumina HiSeq 2500) for samples obtained using the human ES cell line (WA09 strain) 12 single cell.
  • HiSeq 2500 was run at an installed concentration of 6 pM. After obtaining the array, data processing was performed according to the following flow and illustrated (FIG. 5). After mapping the sequence data to the human reference genome sequence (masked by GRCh38 release 85, N) downloaded from the Ensembl database using the Bismark command of the Bismark software (see the method of Guo et al.), Further reference using the bimark_methylation_extractor command The position information of the CpG site on the genome sequence was acquired and illustrated.
  • the commands and options at the time of execution of bismark and bismark_methylation_extractor are as follows. In order to execute the Bismark software, the Bowtie 2 software and the SAMtools software need to be installed.
  • the program for drawing the Lorentz curve and calculating the Gini coefficient was implemented using the R language (Fig. 6).
  • a Y-shaped adapter containing a biotinylated barcode on both sides of the following sequence was used as an adapter.
  • a Y-shaped adapter was prepared by annealing a universal adapter with a biotinylated 5 ′ end and an index adapter with a biotinylated 3 ′ end.
  • the sequence of the universal adapter is as follows.
  • samples II, III, IV 18 ⁇ L of samples (Samples II, III, IV) were subjected to bisulfite treatment. Nuclease-free water was added to the sample to adjust to 25 ⁇ L. 125 ⁇ L of CT conversion buffer was added and mixed well. Bisulfite treatment was performed at 98 ° C. for 10 minutes, at 64 ° C. for 2 hours, and then at 4 ° C. The treated DNA was centrifuged after adding 1 ⁇ l of tRNA (10 ng / ⁇ l) to the binding buffer using a methylcode bisulfite conversion kit (ThermoFisher SCIENTIFIC product number MECOV-50). The final elution used 25 ⁇ L of elution buffer preheated at 50 ° C. The amount was measured and divided into three equal parts (newly sample II (BS (+) Res (-) in FIG. 7), sample III (BS (+) Res (+) in FIG. 7), sample IV (BS in FIG. 7) (+) Res (ATP))).
  • the rescue reaction included four steps 5-1), 5-2), 5-3) and 5-4).
  • the beads were washed twice with 1 ⁇ BWT buffer (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.02% Tween-20), and then EBT buffer (10 mM Tris-HCl (pH 8)). 5), 0.02% Tween-20) and washed once.
  • 1 ⁇ BWT buffer 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.02% Tween-20
  • EBT buffer 10 mM Tris-HCl (pH 8)
  • phosphatase reaction A phosphatase mixture was prepared using 1x buffer and nuclease-free water. The beads of 5-1) were captured and the supernatant was removed, and then suspended in a phosphatase mixture and transferred to a PCR tube. 1 ⁇ L of FastAP Thermosensitive Alkaline Phosphatase (ThermoFisher SCIENTIFIC product number EF0654) was added, reacted at 37 ° C. for 10 minutes, and enzyme-inactivated at 75 ° C. for 5 minutes. After the reaction, it was transferred to a LoBind tube to capture the beads, and washed twice with EBT buffer.
  • FastAP Thermosensitive Alkaline Phosphatase Thermosensitive Alkaline Phosphatase
  • a kinase mixture was prepared using 1x buffer and nuclease-free water. A final concentration of 1 mM ATP was added to the kinase mixture. The beads of 5-2) were captured and the supernatant was removed, and then suspended in a kinase mixture and transferred to a PCR tube. 1 ⁇ L of T4 polynucleotide kinase (TaKaRa product number 2021A) was added, reacted at 37 ° C. for 30 minutes, and enzyme-inactivated at 70 ° C. for 5 minutes. After the reaction, it was transferred to a LoBind tube to capture the beads, and washed twice with EBT buffer.
  • T4 polynucleotide kinase T4 polynucleotide kinase
  • Second round PCR enrichment A PCR mixture was created as follows. 1 ⁇ Phusion HF PCR master mix with HF buffer (see Guo et al. Literature), 500 nM primer (see Guo et al. Literature), and sample. Thereafter, the PCR reaction was performed as follows. Set at 98 ° C. for 2 minutes ⁇ 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute 22 cycles ⁇ 72 ° C. for 5 minutes ⁇ 4 ° C. After completion of the PCR reaction, the PCR product was purified once with 1: 1 AMPure beads, eluted with 25 uL of nuclease-free water and collected.
  • Quantitative PCR was performed using a human-specific gene primer (Alu3 primer sequence) using the sample after gel cutting (250-700 bp) and purification as a template.
  • the primer sequences used are as follows. 5'-TGGAAGACCAGTAGGATGATTG-3 '(SEQ ID NO: 20) 5'-GTCTTACCATGTATCTCTGTGC-3 '(SEQ ID NO: 21)
  • the present invention methylation analysis of a small amount of DNA sample such as DNA derived from a single cell becomes possible. Therefore, the present invention has high utility value in fields such as medicine, pharmacy, biology, genetics, and agriculture.
  • SEQ ID NO: 1 is the base sequence of the adapter used in the examples.
  • the 5 'end is biotinylated and the cytosine is 5-methyl-dC.
  • SEQ ID NO: 2 is the base sequence of the adapter used in the examples.
  • the 3 'end is biotinylated and the cytosine is 5-methyl-dC.
  • SEQ ID NO: 3 is the base sequence of the PCR amplification primer used in the examples.
  • SEQ ID NO: 4 is the base sequence of the universal adapter used in the examples.
  • the 5 ′ end is biotinylated, cytosine is 5-methyl-dC, and the 3 ′ end CT is phosphorothioated.
  • SEQ ID NO: 5 is the base sequence of the index adapter used in the examples.
  • the 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 6 is the base sequence of the primer for quantitative PCR used in the examples.
  • SEQ ID NO: 7 is the base sequence of the primer for quantitative PCR used in the examples.
  • SEQ ID NO: 8 is the base sequence of the index adapter used in the examples.
  • the 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 9 is the base sequence of the index adapter used in the examples.
  • the 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 10 is the base sequence of the index adapter used in the examples.
  • the 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 11 is the base sequence of the index adapter used in the examples.
  • the 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 12 is the base sequence of the index adapter used in the examples.
  • the 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 13 is the base sequence of the index adapter used in the examples.
  • the 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 14 is the base sequence of the index adapter used in the examples. The 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 15 is the base sequence of the index adapter used in the examples. The 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 16 is the base sequence of the index adapter used in the examples.
  • the 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 17 is the base sequence of the index adapter used in the examples. The 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 18 is the base sequence of the index adapter used in the examples. The 5 'end is phosphorylated, cytosine is 5-methyl-dC, and the 3' end is biotinylated.
  • SEQ ID NO: 19 is the base sequence of the index adapter used in the examples.
  • SEQ ID NO: 20 is the base sequence of the primer for quantitative PCR used in the examples.
  • SEQ ID NO: 21 is the base sequence of the primer for quantitative PCR used in the examples.

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Abstract

La présente invention concerne un procédé pour une analyse de méthylation d'ADN utilisant un traitement au dioxyde de soufre, ledit procédé étant caractérisé en ce que le rendement de l'échantillon d'ADN qui peut être amplifié par une réaction en chaîne par polymérase après traitement au dioxyde de soufre est augmenté en traitant l'échantillon d'ADN après un traitement au dioxyde de soufre avec une ligase d'ADN monocaténaire.
PCT/JP2016/087100 2015-12-14 2016-12-13 Procédé de préparation d'échantillon pour analyse de méthylation d'adn Ceased WO2017104675A1 (fr)

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Citations (1)

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JP2007525963A (ja) * 2003-06-20 2007-09-13 イルミナ インコーポレイテッド 全ゲノム増幅および遺伝型決定のための方法および組成物

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WO2009131223A1 (fr) * 2008-04-25 2009-10-29 地方独立行政法人東京都健康長寿医療センター Procédé pour l'analyse de méthylation d'adn
EP2682478A1 (fr) * 2012-07-02 2014-01-08 Institut Curie Procédés et dispositifs pour détecter des macro-ions dans un milieu liquide

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007525963A (ja) * 2003-06-20 2007-09-13 イルミナ インコーポレイテッド 全ゲノム増幅および遺伝型決定のための方法および組成物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GUO H. ET AL.: "Single- cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing", GENOME RES., vol. 23, no. 12, 2013, pages 2126 - 2135, XP055393268 *

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