WORKFLOW GUIDE Epigentics and DNA Methylation Advances in Capillary Electrophoresis-Based Methods for DNA Methylation Analysis Me DNA Methylation Methyl groups added to specific DNA bases repress gene activity. C G Me G C C G Me Histone Histone Modification Many different modifications to histones, including methylation and acetylation, have been identified. These modifications can alter the activity of the DNA wrapped around them. Chromosome Figure 1. The two main components of the epigenetic code, DNA methylation and histone modification Epigenetics and DNA Methylation been associated with many human diseases, including cancer. The term ‘Epigenetics’ describes heritable genetic modifications Patterns of DNA methylation are set during embryogenesis that are not attributable to changes in the primary DNA and re-established during early development by DNA sequence. Epigenetic modifications play a key role in regulating methyltransferase and demethylase enzymes. ‘CpG islands’ gene expression, and therefore are critical to the development, (cytosine-phospho-guanine) are 300-3000 base pair stretches regulation and maintenance of the normal cell. There are of DNA that are CpG rich. CpG islands are often located in three inter-related forms of epigenetic inheritance: genomic the promoter regions of genes where in the normal cell they imprinting, DNA methylation and histone modification. Of these, are typically unmethylated, thus allowing transcription. In DNA methylation is the most well-characterized epigenetic contrast, CpGs found outside promoter regions are commonly modification and forms the focus of this guide. methylated, and are believed to be responsible for silencing the transcription of repetitive sequences and parasitic DNA methylation is involved in the regulation of many sequence elements, such as viral DNA. Consequently, cellular processes, including X chromosome inactivation, aberrant methylation can lead to either silencing of critical chromosome stability, chromatin structure, embryonic genes or increased expression of detrimental factors. development and transcription. Aberrant DNA methylation has CYTOSINE Metabolic Diseases Cardio- vascular Expression Diseases METHYLATION Auto- Regulation immune Diseases Cancer T C T C T METHYLCYTOSINE Figure 2. DNA methylation inhibits gene expression and aberrant methylation has been implicated in many diseases includingA cancerT A G C DNA Methylation Analysis analysis techniques of bisulfite treated DNA include fragment HSO-3 As an epigenetic event, methylation is not preserved analysis using a methylation sensitive mobility shift assay, T U T C G during amplification processes such as PCR or whole gene which yields a cumulative answer for all CpGs across an amplification (WGA). Early methods for detecting and entire amplicon and Single Base Extension, which provides measuring DNA methylation relied on Southern blot-based quantitative information on theA methylationA A stateG ofC an approaches using methylation sensitive enzymes. These individual CpG. The following pages illustrate the typical methods, however, have many drawbacks: they require large workflow for methylation analysis, highlighting technical amounts of high quality DNA, incomplete restriction digests considerations for each step and describing the benefits are frequent, and they do not target specific regions of of Applied Biosystems solutions for analysis of bisulfite interest. Bisulfite treatment of DNA transforms an epigenetic converted DNA. event to a genetic change which is then able to be analyzed using PCR-based methods. Bisulfite deaminates non- T C T C T methylated cytosine residues to uracil, leaving methylated cytosines unchanged. After PCR, the uracils are converted A T A G C to thymine while the methyl Cs are converted to regular Cs. The sample can then be compared to a non-bisulfite treated HSO-3 sample to identify the methylated sites. T U T C G Various technologies may be used to analyze bisufite treated DNA. Microarray and next generation sequencing technologies allow for macro-level identification of methylation A A A G C patterns, providing clues as to which regions should be further elucidated. After identification of candidate regions, capillary PCR electrophoresis (CE)-based sequencing is used for validation T T T C G and remains the gold standard in validating DNA methylation results. CE sequencing provides detailed information for A A A G C each CpG in the entire amplicon and allele-specific haplotype information may be enabled through cloning. Additional Figure 3. Bisulfite conversion of unmethylated cytosine followed by PCR 1 2 3 4 5 DNA Extraction Bisulfite Conversion Primer Design & Detection Data Analysis Amplification T C T C T A T A G C HSO DNA Methylation Workflow DNA Extraction -3 T U T C G A A A G C Description DNA is isolated from various sample types including blood, cultured cells, and tissue (fresh/frozen and formali-fixed-paraffin embedded FFPE). Technical Considerations • DNA purity is critical as contaminating protein inhibits bisulfite conversion and PCR • FFPE samples are difficult to analyze due to variation in DNA quantity, quality and purity AB Solutions Single Tube to 96 Samples - MELT™ Total Nucleic Acid Isolation for Blood and Cell Cultures - RecoverALL™ Total Nucleic Acid Isolation from FFPE tissue Automated Sample Prep - NucPrep® DNA Isolation Kit - BloodPrep® DNA Isolation Kit Benefits • Multiple kits for DNA isolation—Applied Biosystems provides several nucleic acid extraction kits to meet a variety of sample type and throughput needs • High-quality, high-molecular weight DNA—free from inhibitors of PCR and suitable for any DNA-based assay • High-quality DNA from FFPE samples—RecoverAll Kit is optimized to release a maximal amount of RNA and DNA fragments of all sizes with typical yields of >50% that of unfixed tissue from the same sample source 120 ed 100 80 60 40 % Samples successfully amplifi 20 0 RARB GSTP PTEN e2f2 raldgs SDCC APC Target RecoverAll Competitor Q Figure 4. Amplification success rate of bisulfite-modified FFPE-extracted DNA using RecoverAll vs. Competitor Q Kit 1 1 2 32 34 45 5 DNA Extraction DNABisulfite Extraction Conversion BisulfitePrimer DesignConversion & PrimerDetection Design & DataDetection Analysis Data Analysis Amplification Amplification T C T C T T C T C T A T A G C A T A G C HSO HSO-3 Bisulfite Conversion -3 Primer Design & T U T C G AmplificationT U T C G A A A G C A A A G C DNA is isolated from various sample types including blood, cultured cells, DNA is treated with bisulfite which deaminates unmethylated cytosines Primers are designed to amplify the regions of interest and PCR amplified. and tissue (fresh/frozen and formali-fixed-paraffin embedded FFPE). to Uracil. • DNA purity is critical as contaminating protein inhibits bisulfite conversion • Resin-based purification methods have higher affinity for methylated • Primer design is critical as the converted DNA is difficult to amplify due and PCR DNA, resulting in sample bias to the less complex, 3-base genome - Different Tm calculation • FFPE samples are difficult to analyze due to variation in DNA quantity, • Harsh denaturation processes fragment DNA - Increased non-specific priming quality and purity • Incomplete bisulfite conversion leads to false positives • Inefficient purification and recovery leads to poor yields • Long term stability of bisulfite converted DNA at 4°C Single Tube to 96 Samples Bisulfite Conversion Primer Design - MELT™ Total Nucleic Acid Isolation for Blood and Cell Cultures - methylSEQr™ Bisulfite Conversion Kit - Methyl Primer Express® Software - RecoverALL™ Total Nucleic Acid Isolation from FFPE tissue Automated Sample Prep Amplification - NucPrep® DNA Isolation Kit - Thermocycler Systems - BloodPrep® DNA Isolation Kit - AmpliTaq Gold® Kit • Multiple kits for DNA isolation—Applied Biosystems provides several nucleic • Unbiased DNA recovery—methylSEQr™ Kit enables more accurate • Less PCR failure—Methyl Primer Express® Software algorithm is acid extraction kits to meet a variety of sample type and throughput needs downstream quantitative analysis optimized for low complexity DNA, providing rapid primer design with • High-quality, high-molecular weight DNA—free from inhibitors of PCR and less PCR failure suitable for any DNA-based assay • Freely available primer design software—Methyl Primer Express® • High-quality DNA from FFPE samples—RecoverAll Kit is optimized to Software can be downloaded at www.appliedbiosystems.com/ release a maximal amount of RNA and DNA fragments of all sizes with • Optimize PCR conditions for difficult sequences—with the added control typical yields of >50% that of unfixed tissue from the same sample source of VeriFlex™ Blocks, you have six independent temperature blocks providing precise control over your PCR optimization Figure 5. Peak obtained using methylSEQr Kit demonstrates unbiased recovery of methylated and unmethylated fragments whereas peak height for unmethylated DNA from the same sample is significantly reduced with Competitor Q Kit • Less fragmentation—methylSEQr provides more high-quality DNA for better amplification • Higher Yields—methylSEQr provides more high-quality DNA available from minimal starting material • Increased sample stability—methylSEQr Kit converted DNA is stable for up Figure 6: Veriti™ 96-Well Thermal Cycler provides independent temperature to 3 years at 4°C blocks for PCR optimization For more information visit: www.appliedbiosystems.com/methylseqr 1 2 1 3 2 4 3 5 4 5 DNA Extraction