EZ DNA甲基化试剂盒-Gold

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EZ DNA Methylation-Gold Kit 说明书

EZ DNA Methylation-Gold™ KitCatalog Nos. D5005 & D5006Highlights•Complete bisulfite conversion of GC-rich DNA in less than 3 hours.• A coupled heat denaturation/conversion reaction step streamlines the conversion of unmethylated cytosines into uracil.•DNA precipitations are omitted. Instead, DNA is cleaned and desulphonated in a single step using state-of-the-art spin columns.•Eluted, ultra-pure DNA is ideal for use in subsequent molecular-based analyses.ContentsProduct Contents (1)Introduction to DNA Methylation (2)Product Description (3)Product Specifications (4)Reagent Preparation (4)Protocol (5)Appendix (6)Frequently Asked Questions (7)Ordering Information (8)List of Related Products (9)For Research Use Only Ver.2.1.0Page 1 Product Contents:EZ DNA Methylation-Gold™ Kit D500550 rxns. D5006 200 rxns. Storage Temperature CT Conversion Reagent* 5 Tubes 20 Tubes Room Temp. M-Dilution Buffer 1.5 ml 7 ml Room Temp. M-Dissolving Buffer 500 µl 1.2 ml Room Temp. M-Binding Buffer 30 ml 125 ml Room Temp. M-Wash Buffer**6 ml 24 ml Room Temp. M-Desulphonation Buffer 10 ml 40 ml Room Temp. M-Elution Buffer1 ml 4 ml Room Temp. Zymo-Spin™ IC Columns 50 ct. 200 ct. Room Temp. Collection Tubes 50 ct. 200 ct. Room Temp.Instruction Manual11-Note - Integrity of kit components is guaranteed for one year from date of purchase. Reagents are routinely tested on a lot-to-lot basis to ensure they provide maximal performance and reliability.* 900 µl water, 300 µl M-Dilution Buffer , and 50 µl M-Dissolving Buffer must be added per tube of CT Conversion Reagent prior to use.** Add 24 ml of 100% ethanol to the 6 ml M-Wash Buffer concentrate (D5005) or 96 ml of 100% ethanol to the 24 ml M-Wash Buffer concentrate (D5006) before use.The EZ DNA Methylation-Gold™ Kits are patent pending.The Polymerase Chain Reaction (PCR) process is covered by U.S. Pat. Nos. 4,683,195 and 4,683,202 assigned to Hoffmann-La Roche. Patents pending in other countries. No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of Zymo Research’s EZ DNA Methylation kits. Further information on purchasing licenses to practice the PCR process can be obtained from the director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501.Use of Methylation Specific PCR (MSP) is protected by US Patents 5,786,146 & 6,017,704 & 6,200,756 & 6,265,171 and International Patent WO 97/46705. No license under these patents to use the MSP process is conveyed expressly or by implication to the purchaser by the purchase of this product.Note - ™ Trademarks of Zymo Research Corporation. This product is for research use only and should only be used by trained professionals. Some reagents included with this kit are irritants. Wear protective gloves and eye protection. Follow the safety guidelines and rules enacted by your research institution or facility.Note: Satisfaction of all Zymo Research products is guaranteed. If you should be dissatisfied with this product please call 1-888-882-9682.Page 2Introduction to DNA Methylation:DNA methylation is a naturally occurring event in both prokaryotic and eukaryotic organisms. In prokaryotes DNA methylation provides a way to protect host DNA from digestion by restriction endonucleases that are designed to eliminate foreign DNA, andin higher eukaryotes DNA methylation functions in the regulation/control of gene expression (1). It has been demonstrated that aberrant DNA methylation is a widespread phenomenon in cancer and may be among the earliest changes to occur during oncogenesis (2). DNA methylation has also been shown to play a central role in gene imprinting, embryonic development, X-chromosome gene silencing, and cell cycle regulation. In many plants and animals, DNA methylation consists of the addition of a methyl group to the fifth carbon position of the cytosine pyrimidine ring via a methyltransferase enzyme (3). The majority of DNA methylation in mammals occurs in 5’-CpG-3’ dinucleotides, but other methylation patterns do exist. In fact, about 80 percent of all 5’-CpG-3’ dinucleotides in mammalian genomes are found to be methylated, whereas the majority of the twenty percent that remain unmethylated are within promoters or in the first exons of genes. The ability to detect and quantify DNA methylation efficiently and accurately has become essential for the study of cancer, gene expression, genetic diseases, as well as many other important aspects of biology. To date, a number of methods have been developed to detect/quantify DNA methylation including: high-performance capillary electrophoresis (4) and methylation-sensitive arbitrarily primed PCR (5). However, the most common technique used today remains the bisulfite conversion method (6). This technique involves treating methylated DNA with bisulfite, which converts unmethylated cytosines into uracil. Methylated cytosines remain unchanged during the treatment. Once converted, the methylation profile of the DNA can be determined by PCR amplification followed by DNA sequencing (see below).DNA sequencing results following bisulfite treatment. DNA withmethylated C mpG at nucleotide position #5 was processed using the EZ DNA Methylation™ Kit . The recovered DNA was amplified by PCR and then sequenced directly. The methylated cytosine at position #5 remained intact while the unmethylated cytosines at positions #7, 9, 11, 14 and 15 were completely converted into uracil following bisulfite treatment and detected as thymine following PCR.References:1. Costello JF, Plass CJ. Med. Genet. 2001; 38(5): 285-303.2. Stirzaker C. Cancer Res. 1997; 57(11): 2229-2237.3. Adams RL. Bioessays. 1995; 17(2): 139-145.4. Fraga MF, et al . Electrophoresis. 2000; 21(14): 2990-2994.5. Gonzalgo ML. Cancer Res. 1997; 57(4): 594-599.6. Frommer M. Proc. Natl. Acad. Sci. USA. 1992; 89(5): 1827-1831.Page 3 Product Description:The EZ DNA Methylation-Gold TM Kit is a refinement of our popular EZ DNA Methylation™ Kit . The EZ DNA Methylation-Gold TM Kit integrates DNA denaturation and bisulfite conversion processes into one-step. This is accomplished using temperature denaturation to replace chemical denaturation with sodium hydroxide in the previous protocol. Also, the kit has been streamlined for high yield recovery of DNA following DNA bisulfite conversion. Both kits are based on a three-step reaction process between cytosine and sodium bisulfite resulting in cytosine being converted into uracil. The EZ DNA Methylation-Gold™ and EZ DNA Methylation™ Kits share innovative in-column desulphonation technology that eliminates cumbersome DNA precipitation steps while providing researchers consistent results every time. The kits have been designed to minimize template degradation, loss of DNA during treatment and clean-up, and to provide complete conversion of unmethylated cytosines. Recovered DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion, sequencing, microarrays, etc . An outline comparing the EZ DNA Methylation-Gold™ Kit procedure to Zymo Research’s other methylation kits is shown below.Outline of the EZ DNA Methylation™, EZ DNA Methylation-Gold™ and EZ DNA Methylation-Direct™ Kit procedures.Selected EZ DNA Methylation™ Kit Citations: 1. Ehrich M, et al. Nuc. Acids Res. 2007; 35 (5): e29 2. Kaneda M, et al. Nature. 2004; 429: 900-903 3. Zhang F, et al. Proc. Natl. Acad. Sci. USA. 2007; 104 (11): 4395-4400. 4. Oda M, et al. Genes & Dev. 2006; 20: 3382-3394. 5. England RPM, et al. Nature Meth. 2005; 2: 1-2.Page 4Specifications:•DNA Input: Samples containing 500 pg - 2 µg of DNA. For optimal results, the amount of input DNA should be from 200 to 500 ng.•Conversion Efficiency: > 99% of non-methylated C residues are converted to U; > 99% protection of methylated cytosines.•DNA Recovery: > 75%Reagent Preparation:•Preparation of CT Conversion ReagentThe CT Conversion Reagent supplied within this kit is a solid mixture and must beprepared prior to first use. Prepare as follows:1. Add 900 µl water, 300 µl of M-Dilution Buffer, and 50 µl M-Dissolving Buffer toa tube of CT Conversion Reagent.2. Mix at room temperature with frequent vortexing or shaking for 10 minutes.Note: It is normal to see trace amounts of undissolved reagent in the CT Conversion Reagent.Each tube of CT Conversion Reagent is designed for 10 separate DNA treatments.Storage: The CT Conversion Reagent is light sensitive, so minimize its exposure to light. Forbest results, the CT Conversion Reagent should be used immediately following preparation. If notused immediately, the CT Conversion Reagent solution can be stored overnight at roomtemperature, one week at 4°C, or up to one month at -20°C. Stored CT Conversion Reagentsolution must be warmed to 37°C, then vortexed prior to use.•Preparation of M-Wash BufferAdd 24 ml of 100% ethanol to the 6 ml M-Wash Buffer concentrate (D5005) or 96 mlof 100% ethanol to the 24 ml M-Wash Buffer concentrate (D5006) before use.Page 5 Protocol:1. Add 130 µl of the CT Conversion Reagent to 20 µl of your DNA sample in a PCRtube. If the volume of the DNA sample is less than 20 µl, make up the difference with water. Mix the sample by flicking the tube or pipetting the sample up and down, then centrifuge the liquid to the bottom of the tube.2. Place the sample tube in a thermal cycler and perform the following steps*:1. 98°C for 10 minutes2. 64°C for 2.5 hours3. 4°C storage up to 20 hours.*For some samples, alternative parameters may yield improved results (see Appendix). If you havebeen using this kit with good results using different reaction conditions than described above, you can continue using those same conditions.3. Add 600 µl of M-Binding Buffer to a Zymo-Spin™ IC Column and place thecolumn into a provided Collection Tube .4. Load the sample (from Step 2) into the Zymo-Spin™ IC Column containing the M-Binding Buffer . Close the cap and mix by inverting the column several times.5. Centrifuge at full speed (>10,000 x g ) for 30 seconds. Discard the flow-through.6. Add 100 µl of M-Wash Buffer to the column. Centrifuge at full speed for 30seconds.7. Add 200 µl of M-Desulphonation Buffer to the column and let stand at roomtemperature (20°C – 30°C) for 15 - 20 minutes. After the incubation, centrifuge at full speed for 30 seconds.8. Add 200 µl of M-Wash Buffer to the column. Centrifuge at full speed for 30seconds. Add another 200 µl of M-Wash Buffer and centrifuge for an additional 30 seconds.9. Place the column into a 1.5 ml microcentrifuge tube. Add 10 µl of M-Elution Bufferdirectly to the column matrix. Centrifuge for 30 seconds at full speed to elute the DNA.The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. For long term storage, store at or below -70°C. We recommend using 1 - 4 µl of eluted DNA for each PCR, however, up to 10 µl can be used if necessary. The elution volume can be > 10 µl depending on the requirements of your experiments, but small elution volumes will yield more concentrated DNA.Note: For DNA volumes >20 µl , an adjustment needs to be made during the preparation of the CT Conversion Reagent . The amount of water is decreased 100 µl for each 10 µl increase in DNA sample volume. For example, for a 40 µl DNA sample, 700 µl of water is added to make the CT Conversion Reagent . The maximum DNA sample volume to be used for each conversion reaction is 50 µl. Do not adjust the volumes of either the M-Dissolving Buffer or M-Dilution Buffer . The capacity of the collection tube with the column inserted is 800 µl. Empty the collection tube whenever necessary to prevent contamination of the column contents by the flow-through. Alternatively, water or TE (pH ≥ 6.0) can be used for elution if required for your experiments.Page 6 Appendix: Bisulfite Conversion and PCR Optimization1. Reaction Conditions: The reaction conditions given in Step 2 of the Protocol will generateconsistent results for both easy and difficult to convert template DNAs including those thatare GC rich. However, the two protocols provided below (alternative 1 & 2) may yield betterresults in PCR amplification of longer DNA fragments. However, should the DNA templatehave >80% GC composition, then these conditions may result in incomplete templatecytosine to uracil conversion.Alternative 1:1. 98°C for 10 minutes2. 53°C for 30 minutes3. 53°C for 6 minutes}8 cycles4. 37°C for 30 minutes5. 4°C storageAlternative 2:1. 98°C for 10 minutes2. 53°C for 4 hours3. 4°C storage2. PCR Primer Design. Generally, primers of 24 to 32 bases are required for amplification ofbisulfite converted DNA. For most eukaryotes, all non-methylated cytosine residues will beconverted into uracil during the bisulfite treatment. These Cs should be treated as Ts forprimer design purposes. For example, for the sequence 5’-AACCTTACAGGCAC-3’, thecorresponding primer should be 5’-AA TT TTA T AGG T A T-3’.If the primer contains CpG dinucleotides with uncertain methylation status, then mixed baseswith C and T can be used. Usually, there should be no more than three mixed positions perprimer and they should be located toward the 5’ end of the primer. It is not recommended tohave mixed bases located at the 3’ end of the primer.3.Amount of DNA Required for Bisulfite Conversion. The minimal amount of human ormouse genomic DNA required for bisulfite treatment and subsequent PCR amplification is500 pg. The optimal amount of DNA per bisulfite treatment is 200 to 500 ng. Although, up to2 μg of DNA can also be processed, it should be noted that high input levels of DNA mayresult in incomplete bisulfite conversion for some GC-rich regions.4. PCR Conditions. Usually, 35 to 40 cycles are required for successful PCR amplification ofbisulfite converted DNA. Optimal amplicon size should be between 150 - 300 bp; howeverlarger amplicons (up to 1 kb) can be generated with optimization of the bisulfite reaction andPCR conditions. We have found that annealing temperatures between 55 - 60°C typicallywork well. As most non-methylated cytosine residues are converted into uracil, the bisulfite-treated DNA usually is AT-rich and has low GC composition. Thus, it may be necessary toreduce the annealing temperature accordingly.Non-specific PCR amplification is relatively common with bisulfite treated DNA due to its AT-rich nature. PCR using “Hot-Start” polymerases is strongly recommended for theamplification of bisulfite-treated DNA.5. Quantifying Bisulfite Treated DNA. Following bisulfite treatment of genomic DNA, non-methylated cytosine residues are converted into uracil. The recovered DNA is typically A, U,and T-rich. The original base-pairing no longer exists. Instead, it is single stranded withlimited non-specific base-pairing at room temperature. The absorption coefficient at 260 nmresembles that of RNA. Use a value of 40 µg/ml for Ab260 = 1.0 when determining theconcentration of the recovered bisulfite-treated DNA.Page 7 Frequently Asked Questions:Q: Should the input DNA be dissolved in TE, water, or some other buffer prior toits conversion?A: Water, TE or modified TE buffers can be used to dissolve the DNA and do notinterfere with the conversion process.Q: At what temperature and for how long can converted DNA be stored?A: The sample should be stored at ≤ -20ºC whenever possible. The quality of the DNAshould remain relatively unchanged for up to 3 months.Q: Which Taq polymerase(s) do you recommend for PCR amplification ofconverted DNA?A: We recommend any “Hot-Start” DNA polymerase.Q: Why are there two different catalog numbers for the EZ-96 DNA Methylation-Gold™ Kit?A: The two different catalog numbers are used to differentiate between the bindingplates that are included in the kit. Deep and shallow-well binding plates are available to accommodate most rotors and microplate carriers. Below is a comparison of the two binding plates.Binding Plate Silicon-A™ Plate Zymo-Spin™ I-96 Plate StyleShallow-Well Deep-WellHeight of Binding Plate19 mm (0.75 inches) 35 mm (1.38 inches) Binding Plate/Collection Plate Assembly 43 mm (1.69 inches) 60 mm (2.36 inches) Binding Cap./Minimum Elution Volume 5 µg/30 µl 5 µg/15 µl Catalog NumbersD5007D5008Page 8 Ordering Information:Product Description Catalog No. Kit SizeEZ DNA Methylation-Gold™ Kit D5005 50 rxns.EZ DNA Methylation-Gold™ Kit D5006 200rxns.EZ-96 DNA Methylation-Gold™ Kit (Shallow-Well)D5007 2 x 96 rxns.EZ-96 DNA Methylation-Gold™ Kit (Deep-Well)D5008 2 x 96 rxns.For Individual Sale Catalog No. Amount(s)CT Conversion Reagent D5001-1D5003-11 tube1 bottleM-Dilution Buffer D5005-2D5006-21.5 ml7 mlM-Binding Buffer D5005-3D5006-330 ml125 mlM-Wash Buffer D5001-4D5002-4D5007-46 ml24 ml36 mlM-Desulphonation Buffer D5001-5D5002-510 ml40 mlM-Elution Buffer D5001-6D5002-61 ml4 mlM-Dissolving Buffer D5005-6D5006-6500 µl1.2 mlZymo-Spin™ IC Columns (capped)C1004-50C1004-25050 columns250 columnsCollection Tubes C1001-50C1001-500C1001-100050 tubes500 tubes1,000 tubesZymo-Spin™ I-96 Binding Plates C2004 2plates Silicon-A™ Binding Plates C2001 2 platesConversion Plates w/ Pierceable Cover Film C2005 2plates/films Collection Plates C2002 2 platesElution Plates C2003 2 platesPage 9Popular DNA Purification & Analysis Products from Zymo ResearchDNA Clean & Concentrator-5™ Clean & concentrate DNA from any reaction or “crude” preparation in 2 minutes. A 6 µl minimumelution volume allows for highly concentrated DNA. Designed for samples containing up to 5 µgof DNA.5020050200D4003 (uncapped)D4004 (uncapped)D4013 (capped)D4014 (capped)DNA Clean & Concentrator-25™ Clean & concentrate DNA in minutes. 25 µl minimum elution volume allows for highlyconcentrated DNA. Designed for purifying up to 25 µg of DNA.5020050200D4005 (uncapped)D4006 (uncapped)D4033 (capped)D4034 (capped)DNA Clean & Concentrator-100™ Clean & concentrate DNA in minutes. 100 µl minimum elution volume allows for highlyconcentrated DNA. Designed for purifying up to 100 µg of DNA.2550D4029D4030DNA Clean & Concentrator-500™ Clean & concentrate DNA in minutes. 1 ml minimum elution volume allows for highlyconcentrated DNA. Designed for samples containing up to 500 µg of DNA.1020D4031D4032ZR-96 DNAClean & Concentrator-5™Quick (15 minute), high-output recovery of pure DNA from PCR, endonuclease digestions,plasmid preparations, etc. 10-15 µl minimum elution volume allows for highly concentrated DNA.Designed for samples containing up to 5 µg of DNA.2x964x96D4023D4024Zymoclean™Gel DNA Recovery Kit Purify DNA from high and low-melting agarose gels in minutes 50200D4001D4002ZR-96 Zymoclean™ Gel DNA Recovery Kit High-throughput DNA purification from high and low-melting agarose gels. 2x964x96D4021D4022Pinpoint SlideDNA Isolation System™Recover genomic DNA from paraffin-embedded or fresh tissue sections for PCR. Ideal forisolating DNA from clinical tissue samples.50 D3001Zyppy™Plasmid Miniprep Kit Pellet-Free™ plasmid DNA purification in minutes: (alkaline lysis/spin column format for low 30µl elution volume).50100400D4036D4019D4020Zyppy™Plasmid Midiprep Kit Pellet-Free™ plasmid DNA purification in minutes: (alkaline lysis/spin column format and 150 µlminimum elution volume).2550D4025D4026Zyppy™Plasmid Maxiprep Kit High-purity plasmid DNA purification in minutes: (alkaline lysis/spin column format and 2 mlminimum elution volume).1020D4027D4028ZR Genomic DNA I Kit™Genomic DNA isolation from whole blood, tissue culture cells, solid tissue and liquid samples.(Silica bead format is scalable to fit your requirements). 100400D3004D3005ZR Genomic DNA II Kit™Genomic DNA purification from whole blood, tissue culture cells, solid tissue and liquid samples.No requirement for beads or phenol chloroform. 5020050200D3006 (uncapped)D3007 (uncapped)D3024 (capped)D3025 (capped)ZR-96 Genomic DNA Kit™High-output genomic DNA purification from whole blood, tissue culture cells, solid tissue and liquid samples. No requirement for beads or phenol chloroform. 2x964x96D3010D3011ZR Soil Microbe DNA Kit™ Simple, rapid isolation of humic-free, PCR-quality genomic DNA from soil microbes. 50 D6001 ZR Fungal/BacterialDNA Kit™Simple, rapid isolation of PCR-quality genomic DNA from fungi. 50 D6005 ZR Fecal DNA Kit™ Simple, rapid isolation of PCR-quality genomic DNA from feces. 50 D6010ZR Viral DNA Kit™ Isolation of viral DNA from cell-free body fluids or sample mixtures containing cells at concentrations less than 105 cells per ml. 50200D3015D3016ZR-96 Viral DNA Kit™High-output (96-well) isolation of viral DNA from cell-free body fluids or sample mixtures containing cells at concentrations less than 105 cells per ml. 2x964x96D3017D3018EZ DNA Methylation™ Kit Streamlined kit for the conversion of unmethylated cytosines in DNA to uracil via the chemical-denaturation of DNA using our specially designed CT Conversion Reagent™. DNA is thendesulphonated and subsequently cleaned using Fast-Spin column technology. Ultra-purerecovered DNA can be used for PCR and bisulfite sequencing applications. 502002x962x96D5001D5002D5003 (Shallow-well)D5004 (Deep-well)EZ DNA Methylation-Direct™ Kit Bisulfite conversion of DNA directly from cells, tissue, and blood samples. 502002x962x96D5020D5021D5022 (Shallow-well)D5023 (Deep-well)*Bulk quantities are available upon request. Please contact: busdev@ or call 1-888-882-9682 for assistance.。

RRBS二代测序文章13

1Guo, Wei, Zhiming Dong, Jianli Cui, Yanli Guo, Supeng Shen, Xin Guo, and Gang Kuang. “Aberrant Hypermethylation of RASSF2 in Tumors and Peripheral Blood DNA as a Biomarker for Malignant Progression and Poor Prognosis of Esophageal Squamous Cell Carcinoma.”Clinical & Experimental Metastasis, October 19, 2015. doi:10.1007/s10585-015-9759-5.2You, Yan-Jie, Yu-Ping Chen, Xiao-Xuan Zheng, Stephen J. Meltzer, and Hao Zhang. “Aberrant Methylation of the PTPRO Gene in Peripheral Blood as a Potential Biomarker in Esophageal Squamous Cell Carcinoma Patients.”Cancer Letters 315, no. 2 (February 2012): 138–44. doi:10.1016/j.canlet.2011.08.032.3Takahashi, Saori, Isao Suetake, Jan Engelhardt, and Shoji Tajima. “A Novel Method to Analyze 5-Hydroxymethylcytosine in CpG Sequences Using Maintenance DNA Methyltransferase, DNMT1.”FEBS Open Bio 5 (2015): 741–47. doi:10.1016/j.fob.2015.09.003.4Huang, Yiping, Xiaofei Chang, Juna Lee, Yong Gu Cho, Xiaoli Zhong, Il-Seok Park, Jun-Wei Liu, et al. “Cigarette Smoke Induces Promoter Methylation of Single-Stranded DNA-Binding Protein 2 in Human Esophageal Squamous Cell Carcinoma.”International Journal of Cancer 128, no. 10 (May 15, 2011): 2261–73. doi:10.1002/ijc.25569.5Wang, Zhengkai, Chunyan Chen, Lin Wu, Jiong Liu, Youke Lu, and Fangyu Wang. “Circulating SFRP1 Promoter Methylation Status in Gastric Adenocarcinoma and Esophageal Square Cell Carcinoma.”Biomedical Reports, November 17, 2014. doi:10.3892/br.2014.388.6Guo, Yue, Limin Shu, Chengyue Zhang, Zheng-Yuan Su, and Ah-Ng Tony Kong. “Curcumin Inhibits Anchorage-Independent Growth of HT29 Human Colon Cancer Cells by Targeting Epigenetic Restoration of the Tumor Suppressor Gene DLEC1.”Biochemical Pharmacology 94, no. 2 (March 2015): 69–78.doi:10.1016/j.bcp.2015.01.009.7Li, Sulan, Dongli Yue, Xinfeng Chen, Liping Wang, Jieyao Li, Yu Ping, Qun Gao, et al. “Epigenetic Regulation of CD271, a Potential Cancer Stem Cell Marker Associated with Chemoresistance and Metastatic Capacity.”Oncology Reports, October 24, 2014. doi:10.3892/or.2014.3569.8Singh, Virendra, Laishram Chandreshwor Singh, Madavan Vasudevan, Indranil Chattopadhyay, Bibhuti Bhusan Borthakar, Avdhesh Kumar Rai, Rup Kumar Phukan, et al. “Esophageal Cancer Epigenomics and Integrome Analysis of Genome-Wide Methylation and Expression in High Risk Northeast Indian Population.”OMICS: A Journal of Integrative Biology, October 23, 2015. doi:10.1089/omi.2015.0121.9Osakabe, Akihisa, Fumiya Adachi, Yasuhiro Arimura, Kazumitsu Maehara, Yasuyuki Ohkawa, and Hitoshi Kurumizaka. “Influence of DNA Methylation on Positioning and DNA Flexibility of Nucleosomes with Pericentric Satellite DNA.”Open Biology5, no. 10 (October 2015): 150128.doi:10.1098/rsob.150128.10Baba, Y., M. Watanabe, A. Murata, H. Shigaki, K. Miyake, T. Ishimoto, M. Iwatsuki, et al. “LINE-1 Hypomethylation, DNA Copy Number Alterations, and CDK6 Amplification in Esophageal Squamous Cell Carcinoma.”Clinical Cancer Research20, no. 5 (March 1, 2014): 1114–24.doi:10.1158/R-13-1645.11Peng, DunFa, Tian-Ling Hu, Aixiang Jiang, Mary Kay Washington, Christopher A. Moskaluk, Regine Schneider-Stock, and Wael El-Rifai. “Location-Specific Epigenetic Regulation of the Metallothionein 3 Gene in Esophageal Adenocarcinomas.”Edited by Hassan Ashktorab. PLoS ONE 6, no. 7 (July 19, 2011): e22009. doi:10.1371/journal.pone.0022009.12Ye, Xiaobing, Gang Feng, Nanlin Jiao, Chun Pu, Guohai Zhao, and Guoping Sun. “Methylation of DLEC1 Promoter Is a Predictor for Recurrence in Chinese Patients with Gastric Cancer.”Disease Markers 2014 (2014): 1–6. doi:10.1155/2014/804023.13Kuo, I-Ying, Jia-Ming Chang, Shih-Sheng Jiang, Chung-Hsin Chen, I-Shou Chang, Bor-Shyang Sheu, Pei-Jung Lu, Wei-Lun Chang, Wu-Wei Lai, and Yi-Ching Wang. “Prognostic CpG Methylation Biomarkers Identified by Methylation Array in Esophageal Squamous Cell Carcinoma Patients.”International Journal of Medical Sciences 11, no. 8 (2014): 779–87. doi:10.7150/ijms.7405.2015.11.06 ZXY1.完善ESCC的list（见批注）；2.ESCC的文章（指你找到的关键文章）标注出abstract，introduction，method里面的关键句子。

喉癌组织中LCRG1基因启动子的克隆、突变和甲基化检测

喉癌组织中LCRG1基因启动子的克隆、突变和甲基化检测李金花;曾龙武;谢海龙【摘要】目的观察喉癌候选抑瘤基因(laryngeal carcinoma related gene1,LCRG1)启动子的功能.方法 (1)通过5′-缺失突变、瞬时转染与荧光素酶报告基因活性检测,进一步分析与LCRG1基因表达调控有关的最小启动子区域;(2)通过PCR-单链构象多态性(PCR-single-strand conformation polymorphism,PCR-SSCP)技术检测40例喉癌组织中LCRG1基因启动子突变;(3)应用甲基化特异性PCR(methylation specific PCR,MSP)技术检测40例喉癌组织中LCRG1基因启动子甲基化状态;(4)采用RT-PCR技术检测40例喉癌组织中LCRG1基因的表达.结果(1)LCRG1基因最小启动子片段位于转录起始位点上游-169~-57位点;(2)喉癌组织中LCRG1基因启动子无突变;(3)40例喉癌组织中有5.00% LCRG1基因启动子甲基化改变;(4)RT-PCR检测显示40%(16/40)喉癌组织中LCRG1基因表达下调.结论 LCRG1基因最小启动子位于转录起始位点上游-169~-57位点的DNA片段;LCRG1基因启动子甲基化部分改变可能参与该基因的表达调控.【期刊名称】《临床与实验病理学杂志》【年(卷),期】2013(029)008【总页数】6页(P819-824)【关键词】转录调控;最小启动子;瞬时转染;甲基化【作者】李金花;曾龙武;谢海龙【作者单位】杭州师范大学附属医院病理科,杭州310015;南华大学肿瘤研究所,衡阳421000;南华大学肿瘤研究所,衡阳421000;南华大学肿瘤研究所,衡阳421000【正文语种】中文【中图分类】R739.65喉癌是一种严重危害人类健康的恶性肿瘤，其发生、发展过程复杂，涉及大量相关基因结构和表达调控的改变，瘤基因的激活和抑瘤基因的失活起到关键性作用，喉癌的发病分子机制至今仍未清楚[1]。

甲基化实验步骤

MGMT甲基化检测实验步骤一．亚硫酸氢钠处理DNA（将未甲基化的胞嘧啶脱氨基变为尿嘧啶）使用EZ DNA（北京中西远大科技有限公司）甲基化试剂盒处理样本。

二．DNA样本的处理1.取130微升的CT Conversion Reagent和20微升的DNA样本，加入1.5毫升的离心管中。

混合。

2.准备好水浴锅，将离心管放入98℃水浴锅中10分钟，64℃水浴锅中2.5个小时。

（不能立即开始实验的，将样本放入4℃冰箱中，保存。

）3.将离心管中的溶液吸出，加入到Zymo-Spin TM的萃取柱中。

4.取600微升的M-Binding Buffer加入到萃取柱中，摇晃混匀。

5.转速＞10000转/分钟的离心机中，离心30秒。

倒掉液体。

6.取100微升的M-Wash Buffer加入萃取柱，离心30秒，倒掉液体。

7.取200微升的M-Desulphonation Buffer加入萃取柱，等待15~20分钟，使其充分浸润。

然后离心30秒。

8.取200微升的M--Wash Buffer加入离心管中，离心30秒。

重复此步骤一次，随后将收集柱丢弃，萃取柱放入1.5毫升离心管中。

9.取10微升的M-Elution Bufer加入到萃取柱中，离心30秒，萃取柱丢弃，离心管中得到处理好10微升DNA样本。

三．巢式PCR（体系，程序）第一轮PCR：模板（处理后的DNA），引物：MGMT--JF/R ，dd水，PCR MIX Golden Star。

体系为：20微升=模板DNA 4微升+引物2微升+dd水4微升+Golden Star 10微升（一管）反应条件：95℃ 10min95℃ 30s →52℃ 30s → 40个循环72℃ 30s →72℃ 10min4℃ forever第二轮PCR：模板（第一轮PCR的产物），引物：m-MGMT-F/R及u-MGMT-F/R，dd 水，PCR MIX Golden Star。

体系为：20微升=模板DNA 1微升+引物（m-MGMT-F/R）2微升+dd水7微升+Golden Star 10微升（一管）20微升=模板DNA 1微升+引物（u-MGMT-F/R） 2微升+dd水7微升+Golden Star 10微升（一管）反应条件：95℃ 10min95℃ 30s →64℃ 30s → 40个循环72℃ 30s →72℃ 10min4℃ forever四．PAGE电泳检测配置PAGE胶：12%的胶： 100ml 12ml30%丙烯酰胺： 40ml 4.8ml 10xTBE 10ml 1.2ml 10%过硫酸铵 0.7ml 84μl TEMED 35μl 4.2μl H2O 49.3ml 5.9ml。

DNA甲基化

Genomic DNA bisulfite treatment by EZ DNA Methylation-Gold KitDNA input: 750ng;Preparation of CT conversion reagent1.Add 900ul water, 300ul of M-Dilution Buffer, and 50ul M-Dissolving Buffer to a tube of CTconversion reagent.（store in dark）2.Mix at room temperature with frequent vortexing or shaking for 10min.（on ice）Storage: The conversion reagent solution can be stored overnight at room temperature, one week at 4℃, or up to one month at -20℃.Protocol:1.System: 150ul.CT conversion reagent 130 ulDNA sample750ng+water 20ul2.Condition: 50ul/tube98℃for 10min→64℃for 2.5 h→4℃（PCR）Purification:1.Adding 600ul of M-Binding Buffer and bisulfite product to 1.5ml tube mix together (shaking).2.Load the sample into the Zymo-Spin IC Column.3.Centrifuge at full speed (≥10,000×g) for 30s, repeat 2-3 times, then discard the flow-through（reserve the flow-through in another empty tube）.4.Add 100ul of M-Wash Buffer to the column. Centrifuge at full speed (≥10,000×g) for 30s.5.Add 200ul of M-Desulphonation Buffer to the column and let stand at room temperature for15-20 min, centrifuge at full speed (≥10,000×g) for 30s, and then centrifuge again at full speed (≥10,000×g).6.Add 200 ul of M-Wash Buffer to the column, centrifuge at full speed for 30s, add 200 ul ofM-Wash Buffer to the column again. Centrifuge at full speed for an additional 30s.7.Place the column into a new 1.5ml tube, add 10ul of M-Elution Buffer directly to the columnmatrix. Centrifuge at full speed for 30s to elute the DNA (2 times and the last volume is near to 20ul).8.Measure the concentration.（RNA OD）9.Storage at -20℃ or -70℃.NEST PCR for amplification the candidate geneNEST 1:System: 20ulBisulfite product 3.0ulPrimer(+)(-) 1.0uldNTP 0.5ulTaq 0.5ul10×GC Buffer 10.0ulddH2O 5ulCondition:94℃4min→[94℃1min→50℃30s→72℃ 50s (≤1min 1kb≈1s)]→72℃10min→4℃30 cyclesNEST 2: dilute the NEST1 PCR product to 10 times (add 175ul water).System: 20ulNEST1 product 3.0ulPrimer(+)(-) 1.0uldNTP 0.5ulTaq 0.5ul2×GC Buffer 10.0ulddH2O 5ulCondition:94℃4min→[94℃1min→56℃? 30s→72℃ 40s (≤1min 1kb≈1s)]→72℃10min→4℃40 cyclesGel ExtractionRun gel and extract DNA from gel (BBI kit or GenStar)1% Agarose Gel with fresh PBS buffer1.Excise the DNA fragment from the gel with a clean, sharp scalpel. Weigh the gel slice and transfer to a 1.5mL microfuge tube.2.Add 400ul of Binding buffer II for each 100mg of gel weight. Incubate at 50℃-60℃ for 10min and shake occasionally until agarose is completely dissolved.3.Add the above mixture to the EZ-10 column and let stand for 2min to cool down. Centrifuge at 10,000rmp for 2min and discard the flow-through in the tube. (Twice)4.Add 500ul of Wash Solution, and centrifuge at 10,000rmp for 2min. Discard the solution in the tube.5.Repeat step 4. Centrifuge at 10,000rmp for an additional 2min to remove any residual Wash Buffer, and then centrifuge again at 10,000rmp for 2min.6.Place the column in a clean 1.5ml microfuge tube. Add 30-50ul of Elution Buffer to the center of the column and incubate at room temperature for 2min. Centrifuge at 10,000rmp for 2min to elute DNA. (Twice)Note:It is extremely important to add Elution Buffer to the center of the column. Incubating the column at higher temperature (37℃-50 ℃) may slightly increase the yield. Pre-warming the Elution Buffer at 55℃ to 80℃ may also slightly increase elution efficiency.7.Meature the concentration of the product (OD DNA )8.ElectrophoresisRun gel to check the size of target DNA (50ng per sample). 1% Agarose Gel9.Store the purified DNA at -20℃.Ligation (pGEM-T easy System) (冰上)System 10ulStandard ReactionPositiveControlBackgroundControl2×Rapid ligation buffer,T4 DNAligase5.0ul 5.0ul 5.0ulpGEM-T Easy Vector (25ng) 0.5ul 0.5ul 0.5ul PCR product Xul - - Control Insert DNA - 2.0ul - T4 DNA Ligase 1.0ul 1.0ul 1.0ul ddH2O ? 1.5 ul 3.5 ul Final volume of 10.0ul 10.0ul 10.0ul(ng vector ×kb size of insert/kb size of vector) ×3/1=ng of insertX= ng of insert/concentration of DNACondition:（PCR）1.20℃ for 2h.Or2.4℃ 16h.Storage at -20℃Transformation of DH5α competent cellsLB solid medium (per liter)Agar 15gLB 25gAmp(100mg/mL) 1000uLLB liquid medium (per liter )Tryptone 10gYeast extract 5gNaCl 10g(adjust PH to 7.0 with NaOH)IPTG stock solution (0.1M):1.2g IPTG added with water to 50ml final volume. store at 4℃.X-Gal (2ml):100mg 5-bromo-4-chloro-3-indolyl-β-D-galactoside dissolved in 2mlN, N´-dimethyl-formamide(二甲基甲酰胺避光). Store at -20℃.Ampicillin stock solution (100mg/ml): final concentration 100ul/ml1.Prepare LB/ampicillin/IPTG/X-Gal plates. Mix X×100ul IPTG and X×20ul X-Gal togetherthen spread on the plate in which had ampillin already (1ml LB contain 1ul ampicillin). Stay until the liquid has been absorbed. 打开水浴42℃，预热摇床2.Mix cells by gently flicking the tube then add 20-30ul cells to the 1.5ml tube then add 2-3ulligation product into the same tube (on ice).插到底3.Incubate the tube on ice for 30min.4.Heat-shock the cells for about 45s in a water bath at exactly 42℃, immediately return thetubes to ice for 3min.5.Add 1.0ml room temperature medium (no ampicillin) to the ligation reaction transformations,then incubation for 1.5h at 37℃ with shaking (220rpm).6.Centrifuge at 6000rpm for 2min, leave the medium about 100ul.7.Plate the medium and cells onto duplicate LB/ampicillin/IPTG/X-Gal plates.8.Incubate plates overnight (about 20h) at 37℃.9.Select white single colony into LB medium (600 ul) with ampicillin and shake for 14h. Sequencing。

甲基化实验步骤

一．重亚硫酸盐修饰基因组DNA 的制备采用EZ DNA Mehtylation-Gold Kit TM D5005试剂盒，按照操作说明书分别对提取的Blank 、Cya 基因组DNA 进行重亚硫酸盐修饰。

进行重亚硫酸盐修饰。

1. 分别吸取10μL Blank 和Cya DNA 样品到PCR 管中，加入10μL 水补足20μL ，每个样品加130μL CT conversion reagent 。

轻弹管子或者用枪吹打混匀，离心使液体沉到管底。

底。

2. 将管子放到热循环仪中，运行以下步骤：将管子放到热循环仪中，运行以下步骤：1. 98℃ 10min2. 6464℃℃ 2.5h 2.5h3. 4℃保存可至20h 20h3. 加600μL M-Binding Buffer 到Zymo-Spin TM IC Column, 柱子放在提供的收集管中。

柱子放在提供的收集管中。

4. 把样品（来自步骤2）加到装M-Binding Buffer 的Zymo-Spin TM IC Column 中。

盖上盖子，颠倒混匀。

颠倒混匀。

5. 全速离心（≥10000g ）30s 。

倒掉流穿液。

6.加100μL M-Wash Buffer 到柱子上。

全速离心30s 。

7. 加200μL M-Desulphonation Buffer 到柱子上，室温（到柱子上，室温（20℃-30-30℃）静置℃）静置15-20min 。

孵育完成后，全速离心30s 。

8. 加200μL M-Wash Buffer 到柱子上。

全速离心30s 。

另加200μL M-Wash Buffer ，再离，再离心30s 。

9. 把柱子放到1.5mL 离心管。

垂直加10μL L M-Elution M-Elution M-Elution buffer buffer 到柱子基质。

全速离心30s 洗脱DNA 。

二． PCR 扩增反应模板：重亚硫酸盐修饰的Blank 、Cya 基因组DNA 引物引物上游：CTCAAGCTTCGAA TTTTTTTA TAAGAAGAAGA TA TAG 下游：TAGA TCCGGTGGA TCAAAACTCCACCACAAACCACTT 目的片段长度：476bp 1. 操作步骤：操作步骤：反应体系反应体系模板模板 0.5μL 10×KOD plus buffer 5μL dNTPS Mixture （2.5mM each ） 5μL Mg 2+ 4μL 上游引物上游引物 3μL 下游引物下游引物 3μLDMSO 4μL KOD plus 酶 2μL ddH 2O 23.5μL Total 50μL 反应条件：反应条件：1）95℃ 3min2）95℃ 25s ；48℃ 15s ；72℃ 45s 。

D5020说明书(中文)

北京天漠科技开发有限公司（ZYMO RESEARCH 中国总代理）ZYMO RESEARCHThe Beauty of Science is to Make Things SimpleEZ DNA Methylation-Direct Kit™EZ DNA 甲基化试剂盒- DirectCatalog No. D5020 & D5021操作手册•亚硫酸氢盐直接转化来源于血液，组织或者细胞中的DNA •可很好的兼容小样品量的输入，如10个细胞或50pg的DNA•尤其适用于FFPE（石蜡包埋组织）和LCM（激光捕获显微切割）样品北京天漠科技开发有限公司（ZYMO RESEARCH 中国总代理）EZ DNA Methylation-Direct Kit™产品简述EZ DNA Methylation-direct kit 是EZ DNA Methylation kit 和EZ DNA Methylation-gold kit的改进版，EZ DNA Methylation-direct kit可对来自血液、组织和细胞中的DNA直接进行亚硫酸氢盐转化,而不需要提取或纯化DNA，这个试剂盒的敏感性非常强，可对少至10个细胞或50pg的DNA进行修饰。

此试剂盒与EZ DNA Methylation-gold kit一样都是将DNA变性和转化过程合二为一（见下图），而且在处理和纯化的过程中，可以减少模版降解和DNA的损失，以便完全转化未甲基化的C。

回收的DNA对于PCR扩增，限制性内切酶消化，测序，微阵列等下游操作都是非常理想的。

Page 1试剂制备在使用前添加260μl (D5020)或1040μl (D5021)的Proteinase K Storage Buffer 到Proteinase K 管中。

使得Proteinase K 的终浓度为20 mg/ml，完全溶解后储存在-20°C。

添加790μl的M-Solubilization Buffer和300μl的M-Dilution Buffer到CT Conversion Reagent中混合，然后再添加160μl的M-Reaction Buffer混合。

EZ DNA甲基化试剂盒-Gold

EZ DNA Methylation-Gold KitEZ DNA甲基化试剂盒-GoldCatalog No. D5005Highlights在 3 小时内完全转化富含GC的DNA.两次加热变性的反应步骤简化了由未甲基化的胞嘧啶转化为尿嘧啶的过程.没有DNA 沉淀.并且通过柱层析一步完成DNA的纯化和脱硫.洗脱的超纯DNA 对于一系列的分子生物分析是非常理想的。

描述EZ DNA Methylation-Gold KitTM 是我们EZ DNA Methylation Kit产品的改进版. EZ DNA Methylation-Gold Kit 将DNA 变性过程和亚硫酸氢盐转变过程转化为一步.此步骤采用温度变性来取代以前方案中使用氢氧化钠的化学变性过程. 经过DNA亚硫酸氢盐转变,试剂盒可大量回收到DNA .两种试剂盒都是基于在胞嘧啶与亚硫酸氢钠之间发生的使胞嘧啶转变为尿嘧啶的三步反应. EZ DNA Methylation-Gold 与EZ DNA Methylation Kits 都采用创新的柱内脱磺酸技术来减少不必要的DNA沉淀步骤以便每次都可以提供给研究人员较好的结果.试剂盒的设计可以减少模版降解和纯化过程中的DNA损失, 并且完全转化未甲基化的胞嘧啶.回收的DNA对于PCR扩增、限制性内切酶消化、测序、微阵列等下游分析是很理想的. 下图是EZ DNA Methylation-Gold Kit所呈现出的结果.在经过亚硫酸氢盐处理后的DNA 测序结果。

使用EZ DNA Methylation-Gold Kit处理的甲基化的CmpG (在#5核苷酸位点)DNA。

回收的DNA通过PCR来扩增并且直接测序。

在#5位置上甲基化的胞嘧啶仍然保持完整，当未被甲基化的胞嘧啶(i.e., positions #7, 9, 11, 14 and 15)通过亚硫酸氢盐处理完后全部转化为尿嘧啶。

试剂制备CT Conversion Reagent的制备试剂盒中所提供的CT Conversion Reagent是固体混合物并且一定要在首次使用前制备好. 通过添加900 μl 水, 50 μl 的M-Dissolving Buffer, 和300 μl 的M-Dilution Buffer 到一管的CT Conversion Reagent中. 在室温下溶解并且震荡10分钟或在摇床上摇动10分钟. 如果看到没有溶解的试剂是很正常的, 因为在溶液中的CT Conversion Reagent 已经达到饱和状态.在使用之前将CT Conversion Reagent 溶液在室温(20°C -30°C)下避光保存.每管的CT Conversion Reagent 是针对10次DNA 处理设计的.为了得到较好的结果, CT Conversion Reagent 应当在制备后立即使用.如果不立刻使用的话, CT Conversion Reagent 溶液可以在-20°C存储1星期.而且在使用之前存储的CT Conversion Reagent 溶液一定要在室温下解冻并且通过震动或颠倒2分钟来混合. CT Conversion Reagent 对光很敏感,所以要尽量减少在光下的暴露.M-WASH BUFFER的制备添加24ml (对于200次的D5006应为96ml) 100%的乙醇到M-WASH BUFFER中来制备最终可以使用的M-WASH BUFFERProtocol每次制备的DNA 范围在500 pg 到 2 μg之间. 最佳量为200-500 ng.在PCR管中添加130 μl 的CT Conversion Reagent 到每20 μl DNA 样品中. 如果DNA 样品的体积小于20 μl, 则用水来弥补差量. 通过轻弹试管或移液器操作来混合样品.For Example: 4 μl DNA 样品, 加入16 μl 水, 130 μl CT Conversion ReagentNote: 对于DNA 体积>20 μl, 就要调整CT Conversion Reagent的制备过程. 对于每增加10 μl DNA样品体积来说，水的量就要随之减少100 μl.例如, 40 μl DNA 样品, 就要添加700 μl来制备CT Conversion Reagent. DNA 样品的最大体积为每次转化反应50 μl.不要调整M-Dissolving Buffer 或M-Dilution Buffer的体积.将样品管放到循环变温器并按以下步骤操作:1. 98°C 放置10 分钟2. 64°C放置2.5 小时3. 立刻进行下述操作或者在4°C 下存储(最多20小时).添加600 μl 的M-Binding Buffer 到Zymo-Spin IC Column 中，并将柱放如试剂盒所提供的Collection Tube中.装填样品(从步骤2)到Zymo-Spin IC? Column 含有M-Binding Buffer. 盖上盖将柱颠倒数次来混合样品.全速(>10,000 x g)离心30 秒. 去除流出液.添加200 μl 的M-Wash Buffer 到柱中. 全速离心30 秒.添加200 μl 的M-Desulphonation Buffer 到柱中并且在室温(20 °C- 30 °C) 下放置15-20 分钟. 在培养后, 全速离心30 秒.添加200 μl 的M-Wash Buffer 到柱中. 全速离心30 秒. 再添加200 μl 的M-Wash Buffer 并且离心30 秒.直接添加10 μl 的M-Elution Buffer 到柱基质中. 将柱放置在 1.5 ml 的管中. 全速离心来洗脱DNA.DNA 可立刻进行分析或储存在低于-20°C 下以备以后使用. 我们建议您对于每次PCR使用2 - 4 μl 洗脱的DNA.订购信息产品描述编号试剂规格EZ DNA D5005 50 rxns.Methylation-Gold Kit D5006 200 rxns.。