Advances in Capillary Electrophoresis-Based Methods for DNA Methylation Analysis

WorkFlow Guide Epigentics and DNA Methylation

Me DNA Methylation Methyl groups added to specific DNA bases repress gene activity.

G Me

C C

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

% 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 BisulfiteDNA Conversion Extraction PrimerBisulfite Design Conversion & DetectionPrimer Design & Data AnalysisDetection 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 -3 Detection Data Analysis T U T C G T U T C G

A A A G C A A A G C

Methylation events are detected by sequence or fragment analysis. The particular Results are compared to reference sample of non-methylated DNA. method is determined by the level of resolution and quantitation required. • Sequencing provides detailed information for each CpG within an amplicon • Fragment Analysis yields cumulative information for all CpG’s across an entire amplicon • Single Base Extension provides quantitative results for a particular CpG

• Methylation information across entire CpG island not just one CpG—a • Sequencing algorithms overcompensate for low complexity single CpG may not represent methylation status and capture the resulting 3-base genome and skew data impact on gene expression. May have biologically relevant differential • Limited analysis tools for methylation methylation of CpGs across an island that needs to be characterized. • Quantitative result to understand the %methylation in mixed samples • Bisulfite converted DNA is difficult to sequence

™ Bisulfite Sequencing - Variant Reporter Software ® - BigDye® Terminator Sequencing Kits - SeqScape Software - BigDye® XTerminator™ Purification Kit - Genetic Analysis Systems

Fragment Analysis - GeneMapper® Software v4.0 - GeneScan™ 600 LIZ® Size Standard - Genetic Analysis Systems

Single Base Extension - GeneMapper® Software v4.0 - ABI PRISM® SNaPshot® Multiplex Kit - GeneScan™ 120 LIZ® Size Standard - Genetic Analysis Systems

• More information—sequencing provides detailed information for each CpG • More accurate base calling—KB™ Basecaller algorithm adjusts across an entire amplicon and allele-specific haplotype is enabled via cloning for sequences with skewed base composition in such a way that underrepresented colors are not artificially exaggerated, providing cleaner traces and more accurate methyl C calls

Figure 7. Differential methylation patterns shown over the entire island

• More accurate detection—CE sequencing provides direct detection of DNA methylation without reliance on enzyme or probe affinity • In a sample control of bisulfite conversion—sequencing identifies erroneous data from incomplete bisulfite conversion • Gold Standard Sequencing—highest quality products provide the Figure 8: Comparison of basecalling algorithms performance needed for difficult sequences • Multiple analyses from single reaction—converted DNA can be carried forward into fragment analysis, sequencing and SBE experiments • Fragment analysis is a rapid and inexpensive method to assess the degree of methylation present in a given amplicon References Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions. Kaminsky et.al. Nucleic Bisulfite conversion of genomic DNA for methylation analysis: protocol Acids Res. 2005 Jun 15;33(10):e95 simplification with higher recovery applicable to limited samples and increased throughput. Boyd et.al. Anal Biochem. 2004 Mar 15;326(2):278-80 Resources

Recovery of bisulfite-converted genomic sequences in the methylation-sensitive Methylation Application Information—info.appliedbiosystems.com/methylation QPCR. Munson et.al. Nucleic Acids Research, 2007, 35 (9): 2893-903 Bisulfite Sequencing Manual—http://docs.appliedbiosystems.com/cms/ Methylation-dependent fragment separation: direct detection of DNA groups/mcb_marketing/documents/generaldocuments/cms_039258.pdf methylation by capillary electrophoresis of PCR products from bisulfite- ® converted genomic DNA. Boyd et.al. Anal Biochem. 2006 Jul 15;354 (2): 266-73 Methyl Primer Express Software— www.appliedbiosystems.com/methylprimerexpress

Product Ordering Information

Description Part Number

Sample Prep

MELT™ Total Nucleic Acid Isolation System AM1983

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE AM1975

BloodPrep® DNA Starter Kit 4346860

NucPrep® DNA Isolation Chemistry Starter Kit 4340274

Bisulfite Conversion

methylSEQr™ Bisulfite Conversion Kit 4379580

Primer Design

Methyl Primer Express® Software v1.0 Free download

PCR

Veriti™ 96-Well Thermal Cycler 4375786

AmpliTaq Gold® DNA Polymerase 4311814

Sequencing

BigDye® Terminator v3.1 Cycle Sequencing Kit 4337455

BigDye® Terminator v1.1 Cycle Sequencing Kit 4337450

BigDye® XTerminator™ Purification Kit 4376486

Applied Biosystems 3130xl Genetic Analyzer 3130XL

Single Base Extension

ABI Prism® SNaPshot® Multiplex Kit 4323151

Fragment Analysis

GeneScan™ 600 LIZ® Size Standard 4366589

GeneScan™ 120 LIZ® Size Standard 4324287

Data Analysis

SeqScape® Software v2.5, 45-Day Demo 4327099

GeneMapper® Software v4.0, 30-Day Demo 4366851

Variant Reporter™ Software v1.0, 30-Day Demo 4385270

For Research Use Only. Not for use in diagnostic procedures.

Applera, Applied Biosystems, AB (Design), ABI Prism, BloodPrep, BigDye, GeneMapper, LIZ, NucPrep, Primer Express, SeqScape, and SNaPshot are registered trademarks and GeneScan, KB, methylSEQr, Variant Reporter, VeriFlex Blocks and Veriti are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. MELT and RecoverAll are trademarks of Ambion, Inc. Ambion is a wholly-owned subsidiary of Applera Corporation. All other trademarks are the sole property of their respective owners. © 2007. Applied Biosystems. All rights reserved. Printed in the USA. 08/2007 Publication 106BR14-01

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