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Three Optimized Workflows for Cpg Island Methylation Profiling

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Three Optimized Workflows for Cpg Island Methylation Profiling APPLICATION NOTE Three Optimized Workflows for CpG Island Methylation Profiling Three Optimized Workflows for CpG Island Methylation Profiling DNA methylation is involved in the Discovery regulation of many cellular processes SOLiD™ Do you know your candidate gene? including X chromosome inactivation, No System chromosome stability, chromatin structure, Yes embryonic development, and transcription. Validation/Screening with Capillary Electrophoresis Do you know your candidate region or In mammals only the 5' carbons of the methylation state of that region? cytosines adjacent to guanines (CpG Yes No dinucleotides: cytosine-phospho-guanine) MSMSA Bisulfite Conversion Bisulfite Conversion are substrates for DNA methylation. (MethylSEQr™ Kit) (MethylSEQr™ Kit) Importance of CpG Islands Are there a high number Bisulfite Specific (BSP) CpG islands are 300−3000 base pair of poly T or A, or large Primer Design fragment size (>300 bps)? (Methyl Primer® Express) stretches of DNA that are CG rich, and are often found within unmethylated Yes No regions of promoters. Profiling of CpG Clone based Sequencing Direct PCR Sequencing island methylation indicates that some Fluorescent BSP PCR genes are more frequently methylated Bisulfite Specific (BSP) Primer Design Bisulfite Specific (BSP) Primer Design Data Analysis (Methyl Primer® Express) (Methyl Primer® Express) than others [1]. Hence, CpG islands play a (GeneMapper® Software v. 4.0) critical role in regulating gene expression. CpG islands found outside promoter BSP PCR BSP PCR Identification of a candidate region or the No regions are commonly methylated, and methylation state of Yes that region? are believed to be responsible for silencing Cloning Yes transcription of repetitive sequences Cycle Sequencing Direct PCR Cycle Sequencing Are there a high number and parasitic sequence elements, such of poly T or A, or large No as viral DNA. Aberrant methylation can fragment size (>300 bps)? Data Analysis Data Analysis lead to either silencing of critical genes (Variant Reporter™ Software) (Variant Reporter™ Software) or increased expression of detrimental factors, which has been associated with Figure 1. Decision Tree Outlining the Process for Choosing an Application. When choosing a many human diseases, including cancer methylation workflow, it is necessary to make a clear decision about the research goals first, and then decide [2]. This phenomenon makes the discovery what technical strategy will fit best. of CpG islands and the determination of methods are cumbersome and require 1. Bi s u l fi t e Specific Methylation Sensitive their methylation status the subject of significant optimization. The bisulfite Mobility Shift Assay (Figure 2) intense medical research [3]. conversion method allows precise analysis 2. Bisulfite Specific PCR-based of methylation in a target region by Sequencing (Figure 4) Three Workflows for the Discovery of converting all non-methylated cytosines 3. Bisulfite Specific Cloning-based CpG Islands into uracils, while methylated cytosines Sequencing (Figure 6) Bisulfite DNA conversion is one of remain unchanged. The three workflows the most frequently used techniques described here provide an effective Figure 1 outlines the process to assist in for methylation studies because of solution for methylation analysis with choosing a workflow that is well-suited for its relative simplicity, whereas other straightforward protocols. a specific application. 111951 4pger G7.indd 1 12/22/08 2:40:01 PM The general steps for all three workflows 1 2 3 4 5 are as follows: DNA Extraction Bisulfite Conversion Primer Design & Amplification Fragment Analysis Data Analysis T C T C G Step 1: DNA Extraction A T A G C H2SO4 Prepare high quality genomic template T G T C G A A A G C using RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues, MELT ™ Figure 2. Bisulfite Specific Methylation Sensitive Mobility Shift Assay Workflow. Total Nucleic Acid Isolation System, NucPrep® Chemistry for Tissues, or BloodPrep® DNA Chemistry for Cultured Step 4: Cycle Sequencing and Cleanup Workflow 1: Bisulfite Specific Cells and Blood. For sequencing specific workflows, Methylation Sensitive Mobility perform cycle sequencing of the Shift Assay (MSMSA) Tip: Since purity of gDNA is critical specific target using BigDye Terminator MSMSA is a DNA fragment analysis to the success of complete bisulfite Sequencing Kits. The reactions will then application used to assess the degree of conversion, a proteinase K digestion step require removal of unincorporated dye methylation present in a given amplicon. is recommended to remove all proteins terminators and unused primers using a The first 3 steps in Figure 2 are similar to prior to bisulfite conversion. reliable clean up kit. the general workflow described above. Samples from a fragment analysis assay Step 2: Bisulfite Treatment Tips: For fast and reliable sequencing can also be used in a downstream Use MethylSEQr™ Bisulfite Conversion clean-up, use Big Dye® XTerminator™ Kits sequencing step, making this application Kit to convert non-methylated cytosines to for cleanup of sequencing reactions. ideal for screening. The assay primers are discriminate from methylated cytosines. designed like bisulfite sequencing primers Step 5: Data Collection and Analysis (ie: designed to regions without CpGs) Step 3: Primer Design and Amplification Use Applied Biosystems capillary using Methyl Primer Express Software, so Design primers using Methyl electrophoresis instruments for data that PCR amplification is not dependent Primer Express® Software. For a collection. Perform application specific on methylation status. The forward primer free download of this software, visit analysis of data and interpretation of has a fluorescent dye label (FAM™). www.appliedbiosystems.com/ results to evaluate or confirm the changes The capillary electrophoresis step takes methylprimerexpress. Amplify targets in the methylation status. For secondary advantage of the mobility differences using AmpliTaq Gold® DNA Polymerase analysis of fragment analysis-based between C/T. Hence, methylated and Applied Biosystems Thermal Cyclers. workflows, use GeneMapper® Software C-rich fragments migrate faster than v4.0 or Peak Scanner™ Software (Figure unmethylated T-rich fragments (Figure 3). Tips: Design short amplicons for better 2). For secondary analysis of sequencing- GeneScan™ 600 LIZ® Size Standard is results (150–250 bp for sequencing, 450 based workflows, use Variant Reporter™ used as a sizing marker. For data analysis, bp for cloning). Avoid more than 8 to 9 Software v1.0 or SeqScape® Software v2.6 GeneMapper Software v4.0 or Peak Ts/As in a stretch to reduce slippage. (Figure 4, 6). Scanner Software v1.0 is used. Also, use M13-tailed primers for PCR, so that the same primers can be used for sequencing. CDH1 201 203 205 207 209 211 213 215 217 219 221 223 400 300 Panel A. 100% Methylated. 200 100 0 CDH1 201 203 205 207 209 211 213 215 217 219 221 223 2000 Panel B. 50% Methylated. 1000 0 CDH1 201 203 205 207 209 211 213 215 217 219 221 223 4000 3000 Panel C. 0% Methylated. 2000 1000 0 Figure 3. Cumulative Estimation for Methylation State of CpG Islands within an Amplicon. Methylated fragments migrate faster (Panel A, B) than those that are unmethylated (Panel C). 111951 4pger G7.indd 2 12/22/08 2:40:01 PM 1 2 3 4 5 DNA Extraction Bisulfite Conversion Primer Design & Amplification Sequencing Data Analysis T C T C G A T A G C Reference GTGTCGTGTCGTGTCGTGTC Summary GTGTCGTGTCGTGTCGTGTC H2SO4 T G T C G A A A G C Figure 4. Bisulfite Specific PCR-based Sequencing. 1 2 3 4 5 6 DNA Bisulfite Primer Design Cloning & Sequencing Data Extraction Conversion & Amplification DNA Prep Analysis T C T C G A T A G C H2SO4 T G T C G A A A G C Figure 6. Bisulfite Specific Cloning-based Sequencing. Workflow 2: Bisulfite Specific Workflow 3: Bisulfite Specific For data analysis, Variant Reporter PCR-based Sequencing Cloning-based Sequencing Software v1.0 or SeqScape Software v2.6 This application is used to identify which In this application, a population of is used. CpG dinucleotides are important for the fragments is segregated by cloning, biological process under investigation. providing quantitative results. The first Complete Solution The first 3 steps of the workflow in Figure 3 steps of the workflow in Figure 6 These three methylation profiling 4 are described in the general workflow are described in the general workflow workflows provide a complete solution above. Direct PCR sequencing of a pool of above. This is followed by sequencing from DNA extraction to data analysis fragments takes place providing qualitative and separation of single samples/clones. provided by Applied Biosystems kits, results and information on which CpG Analysis can determine the percentage reagents, instruments, and software. The dinucleotide has changed. Figure 5 of methylation observed for specific user can expect cumulative, qualitative, provides qualitative information for each CpG dinucleotides in a candidate region. and quantitative analysis using these CpG loci across the entire amplicon. For Figure 7 provides quantitative results to workflows. data analysis, Variant Reporter Software understand the percentage of methylation v1.0 or SeqScape Software v2.6 is used. per CpG dinucleotide in mixed samples. References 1. Fe l tu s FA, Lee EK, Costello JF, Plass C, Vertino PM (2003) Predicting aberrant CpG island methylation, Proc Natl Acad Sci 100(21):12253−12258. 2. Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer, Nat Rev Genet 3(6):4z15−428. 3.
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