Practical solutions. for DNA methylation analysis

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Robert Wheeler
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1 DNA Methylation Analysis Tools Practical solutions for DNA methylation analysis Highly efficient and time-saving solutions Locus-specific and whole genome analysis Robust performance

Innovative. Fast. Efficient 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) are common epigenetic modifications of cytosine (C) found in the genomes of many species.

2 Innovative. Fast. Efficient 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) are common epigenetic modifications of cytosine (C) found in the genomes of many species. These modifications have been implicated in numerous biological processes and disease states. 5-mC arises from DNA methyltransferase activity on cytosine and is typically associated with transcriptional down-regulation. 5-hmC results from oxidation of 5-mC and also plays an important role in gene expression. Hence, tools for detecting single 5-mC and 5-hmC modifications are important for a more complete understanding of their biological roles. To meet the needs of scientists working in Epigenetics, we created efficient tools for global and locus-specific analysis of 5-mC, fast systems for detection of 5-hmC within specific DNA loci, highly pure enzymes, and genomic DNA controls for DNA methylation studies. Tools for DNA Methylation Analysis 5-mC analysis Bisulfite conversion kit High efficiency and specificity of the bisulfite conversion reaction in combination with column-based purification of converted DNA Restriction enzyme pairs Specially formulated restriction enzymes for DNA methylation analysis at specific loci CpG Methyltransferase (M.SssI) Fast CpG methyltransferase for in vitro DNA methylation 5-hmC analysis 5-hmC and 5-mC analysis kit 5-hmC analysis and quantification in CCGG sequences T4 β-glucosyltransferase Rapid 5-hmC glucosylation DNA controls CpG Methylated Human Genomic DNA CpG Methylated Jurkat Genomic DNA Jurkat Genomic DNA

Efficient and specific conversion of non-methylated cytosine EpiJET Bisulfite Conversion Kit is designed for simple and reliable bisulfite conversion of DNA for methylation analysis studies.

The purified DNA is ready for downstream analysis in PCR, qpcr, Combined Bisulfite Restriction Analysis (COBRA), and sequencing.

conversion confirmed by COBRA Other vendors FastDigest AluI M C C 4 5 6 7 COBRA analysis of bisulfite converted CpG methylated plasmid DNA.

3 Efficient and specific conversion of non-methylated cytosine EpiJET Bisulfite Conversion Kit is designed for simple and reliable bisulfite conversion of DNA for methylation analysis studies. The kit converts unmethylated cytosines to uracils and allows rapid purification of converted DNA. The purified DNA is ready for downstream analysis in PCR, qpcr, Combined Bisulfite Restriction Analysis (COBRA), and sequencing. Reliable high conversion efficiency and specificity ( 99%) Flexibility two alternative protocols are provided Pure DNA suitable for downstream applications and long-term storage Efficient bisulfite conversion confirmed by COBRA Other vendors FastDigest AluI M C C COBRA analysis of bisulfite converted CpG methylated plasmid DNA. M O GeneRuler Low Range DNA Ladder (Cat #SM), C PCR fragment from bisulfite converted DNA template; C PCR fragment from non-converted DNA template digested with FastDigest AluI (Cat #FD4). Digested PCR fragments from DNA converted using bisulfite conversion kits from (long protocol), (short protocol), -7 other vendors. The absence of additional DNA fragments demonstrates both specific and efficient DNA conversion with the kit. Reproducible results is a fast approach to determine the methylation status of specific loci. Following bisulfite conversion of DNA, restriction sites can be lost or created leading to a different restriction pattern than the one observed in the unconverted control Broad range of input DNA The EpiJET Bisulfite Conversion Kit is specially formulated for efficient conversion of DNA, providing highly reproducible results and robust performance. The EpiJET Bisulfite Conversion Kit is compatible with a broad range of input DNA ranging from 5 pg to µg μg μg ng ng ng pg 5 pg RFU -d(rfy)dt COBRA Temperature, C o Melting curve analysis of PCR fragments from converted and non-converted CpG methylated genomic DNA. Blue PCR products from converted DNA samples using primers specific for converted DNA. Green PCR products from non-converted DNA samples using primers specific for non-converted DNA. Orange PCR products from converted DNA samples using primers specific for non-converted DNA. The absence of PCR products demonstrates the efficiency of DNA conversion. 4 4 Amplification plots of converted DNA samples. Various amounts of CpG Methylated Human Genomic DNA (Cat #SD) were converted with the EpiJET Bisulfite Conversion Kit. A 76 bp genomic DNA fragment was amplified using Maxima SYBR Green qpcr Master Mix (x) (Cat #K5).

Fast analysis of DNA methylation in promoters and gene bodies The EpiJET DNA Methylation Analysis Kit (MspI/HpaII) and EpiJET DNA Methylation Analysis Kit (TaqI/HpyFI) are designed for rapid analysis

4 Fast analysis of DNA methylation in promoters and gene bodies The EpiJET DNA Methylation Analysis Kit (MspI/HpaII) and EpiJET DNA Methylation Analysis Kit (TaqI/HpyFI) are designed for rapid analysis of DNA methylation status at specific loci. The kits include DNA controls and high-quality restriction enzyme izoschizomer pairs with different sensitivity to CpG methylation at 5'-CCGG-' or 5'-TCGA-' sequences. DNA methylation status analysis in promoter and gene body sequences Fast complete digestion of genomic DNA in hour Efficient specially formulated enzymes for genomic DNA methylation analysis Epi HpyFI/Epi TaqI Epi HpaII/Epi MspI.. Methylation in promoter Methylation in gene body Izoschizomers Cq ~. undigested sample methylation sensitive digestion Cq ~.9 completely digested sample Cq ~ RASSF methylation level 5% THRB methylation level % Cq ~ RASSF methylation level % THRB methylation level 85% Efficient detection of methylation status genomic DNA plasmid DNA are restriction endonucleases that recognize the same sequence: MspI/HpaII 5'-CCGG-' TaqI/HpyFI 5'-TCGA-' RASSF promoter THRB gene T m CGA 5% C m CGG % T m CGA % C m CGG 85% Methylation status analysis in various DNA loci. Jurkat Genomic DNA was digested with Epi MspI/Epi HpaII or Epi TaqI/Epi HpyFI enzyme pairs, followed by qpcr analysis using primers for the RASSF promoter or THRB gene. The methylation status of specific loci was determined by analysis of Cq values of analyzed samples. The Cq difference between samples digested with methylation-sensitive enzymes and undigested DNA samples (ΔCq) is shown. Green undigested Jurkat DNA. Orange Jurkat DNA digested with methylation-sensitive restriction enzymes (Epi HpaII or Epi HpyFI). Violet Jurkat DNA digested with methylation-insensitive restriction enzymes (Epi MspI or Epi TaqI). puc9 DNA / Smal mpuc9 DNA / Smal Digestion of methylated and unmethylated DNA using Epi HpyFI and Epi TaqI. Unmethylated and methylated linearized plasmid DNA was mixed with naturally methylated Jurkat genomic DNA and digested for hour with Epi TaqI or Epi HpyFI., 4 undigested DNA,, 5 after digestion with methylation-sensitive Epi HpyFI,, 6 after digestion with methylation-insensitive Epi TaqI. Methylated plasmid DNA and naturally methylated gdna were protected from cleavage with Epi HpyFI, but cleaved with Epi TaqI. Unmethylated plasmid DNA was cleaved with both enzymes. 4

Full assessment of cytosine modification w w w NEW The EpiJET 5-hmC and 5-mC Analysis Kit is an efficient system allowing fast analysis of both 5-hmC and 5-mC in CCGG context.

5 Full assessment of cytosine modification w w w NEW The EpiJET 5-hmC and 5-mC Analysis Kit is an efficient system allowing fast analysis of both 5-hmC and 5-mC in CCGG context. Efficient and reliable accurate quantification of 5-hmC and 5-mC Significantly shorter protocol with enzymes specially formulated for use in Epigenetics Vendor N 5 min -8 h Robust broad range of input DNA Fast no overnight incubation Robust performance Epi MspI/Epi HpaII 4 h 5 min for DNA methylation analysis at 5'-CCGG-' loci 8 T4 β-glucosyltransferase for fast 5-hmC differentiation from 5-mC % of modification 4h 5-hmC conversion step -8 h Quantification of both cytosine modifications by qpcr Full methylation ng ng 5 ng 8 Epi HpaII T4 BGTEpi Mspl Epi Mspl 6 4 mc 5hmc Full hydroxymethylation Cq Cq C Reliable results Cq Cq Cq Cq Cq4 Partial modification Cq < Cq < Cq < Cq4 Cq4 No cytosine modification C % of modification % of modification 4 Different amounts of human brain DNA were glucosylated and digested using EpiJET 5-hmC and 5 mc Analysis Kit, followed by qpcr performed using primers flanking a CCGG region of the VANGLs4 site. Equal percentages of cytosine modification were detected regardless of different DNA input. >4 h Epi HpaII T4 BGTEpi Mspl Epi Mspl 6 Epi MspI/Epi HpaII digestion step Epi HpaII T4 BGTEpi Mspl Epi Mspl ng ng 5 ng Epi HpaII T4 BGTEpi Mspl Epi Mspl mc 5hmc Cq Cq Cq Cq4 Experimental Expected C mc 5hmc 5-hmC, 5-mC and unmodified control DNAs were mixed at the ratio :7:. The EpiJET 5-hmC and 5-mC Analysis Kit was used to analyze the cytosine modifications. The detected percentages matched the expected values. 5

Rapid and efficient enzymes for epigenetics M.

5 µg of linearized plasmid DNA (puc9/smai) was methylated using M.SssI from different suppliers according to vendor recommendations. The methylated plasmid DNA was mixed with.

Methylated plasmid DNA and naturally methylated gdna were protected from cleavage with HpaII and cleaved with MspI.

# EpiJET Bisulfite Conversion Kit 5 rxns K46 EpiJET DNA Methylation Analysis Kit (MspI/HpaII) rxns K44 EpiJET DNA Methylation Analysis Kit (TaqI/HpyFI) rxns K45 EpiJET 5-hmC and 5-mC Analysis Kit 5

SssI) 5 rxns EM8 Jurkat Genomic DNA 5 µg SD CpG Methylated Jurkat Genomic DNA 5 µg SD CpG Methylated Human Genomic DNA 5 µg SD /SciBio Specific selectively transfers glucose to the hydroxymethyl

6 Rapid and efficient enzymes for epigenetics M. SssI w w w T4 β-glucosyltransferase NE W High efficiency complete in vitro methylation of all CpGs Fast reaction is completed in 5 min Stable years at - C Complete CpG methylation in minutes.5 µg of linearized plasmid DNA (puc9/smai) was methylated using M.SssI from different suppliers according to vendor recommendations. The methylated plasmid DNA was mixed with.5 µg of human blood genomic DNA and digested with methylationsensitive HpaII restriction enzyme or methylationinsensitive isoschizomer MspI. Methylated plasmid DNA and naturally methylated gdna were protected from cleavage with HpaII and cleaved with MspI. Vendor B Vendor A genomic DNA plasmid DNA 5 min at 7 C h at C h at 7 C undigested DNA (control) after digestion with HpaII after digestion with MspI Order details Product Size Cat. # EpiJET Bisulfite Conversion Kit 5 rxns K46 EpiJET DNA Methylation Analysis Kit (MspI/HpaII) rxns K44 EpiJET DNA Methylation Analysis Kit (TaqI/HpyFI) rxns K45 EpiJET 5-hmC and 5-mC Analysis Kit 5 rxns K5 T4 β-glucosyltransferase 5 U EO8 CpG Methyltransferase (M.SssI) 5 rxns EM8 Jurkat Genomic DNA 5 µg SD CpG Methylated Jurkat Genomic DNA 5 µg SD CpG Methylated Human Genomic DNA 5 µg SD /SciBio Specific selectively transfers glucose to the hydroxymethyl moiety of 5-hmC Fast complete glucosylation of µg DNA in just 5 min Highly specific and fast glucosylation of 5-hmC Munl BGT M United States Canada Customer Service cs.molbio@thermofisher.com Customer Service cs.molbio@thermofisher.com Technical Support ts.molbio.eu@thermofisher.com Technical Support ts.molbio@thermofisher.com Technical Support ts.molbio@thermofisher.com Tel Fax Tel Fax Tel Fax MB Munl BGT C C C M µg of a fully 5-hydroxymethylated 95 bp PCR fragment was incubated with µl of T4 BGT and compared with performance of BGT from other suppliers (A-C) following recommended protocols. Reaction products were digested with glucosyl-sensitive restriction enzyme MunI. Related products Product Cat. # Phusion U Hot Start DNA Polymerase F-555S/L Luminaris Color HRM Master Mix K/ For further information visit thermoscientific.com/epi 4 Fisher Inc. All rights reserved. All other trademarks are the property of Fisher Inc. and its subsidiaries. Customer Service cs.molbio.eu@thermofisher.com M GeneRuler kb Plus DNA Ladder (#SM4) supplier A, hours supplier B, hour supplier C, min 4, 5 min C-C control Europe

#FD µl (for 200 rxns) Expiry Date: Description. 1 ml of 10X FastDigest Green Buffer. Store at -20 C

#FD µl (for 200 rxns) Expiry Date: Description. 1 ml of 10X FastDigest Green Buffer. Store at -20 C PRODUCT INFORMATION Thermo Scientific FastDigest SalI #FD0644 Lot: 5'...G T C G A C...3' 3'...C A G C T G...5' Supplied with: Store at -20 C 200 µl (for 200 rxns) Expiry Date: BSA included www.thermoscientific.com/onebio