Methylation Analysis Using Methylation‐Sensitive HRM and DNA Sequencing

DNA methylation is a key epigenetic mechanism that regulates gene expression and chromatin structure. This application note describes a workflow for analysis of DNA methylation in specific loci by methylationsensitive high-resolution melting (MS-HRM) followed by DNA sequencing of the reaction products (Figure 1). The workflow relies on Applied Biosystems® real-time PCR and capillary electrophoresis instruments and is supported by a comprehensive set of reagents and software tools. MS-HRM followed by Sanger-based DNA sequencing is a fast, simple method for methylation studies that can be manipulated to provide sensivity for methylation levels as low as 0.1–2%. Procedure Overview MS-HRM analysis offers a simple method for quickly analyzing the methylation status of specific genetic loci. Reaction products that merit further analysis can then be sequenced directly to identify precise methylation patterns. Genomic DNA is treated with bisulfite to convert unmethylated cytosines to uracil, then PCR primers are designed to amplify the region of interest, and the melting profiles of PCR products from the sample DNA are compared to those from fully methylated and unmethylated reference samples. PCR primers and amplicons are designed to provide the level of sensitivity required for the biological question of interest. Samples that are found to warrant further study are sequenced to identify precise methylation patterns. Applied Biosystems provides an integrated set of tools for locus-specific DNA methylation analysis using this optimized, streamlined procedure. The procedure is described in a general way in this application note. For detailed instructions, see the High Resolution Melting Getting Started Guide (P/N 4393102) and the BigDye® Terminator v1.1 Cycle Sequencing Kit Protocol (P/N 4337036). Step 1: Bisulfite Conversion of DNA The first step of the workflow is to treat genomic DNA with bisulfite, which deaminates unmethylated cytosines (C) to form uracil (U), but does not affect methylated cytosines. Thus, bisulfite conversion changes the DNA sequence based on the methylation status of individual nucleotides in genomic DNA, and these changes can be detected via HRM analysis [1]. The Applied Biosystems® methylSEQr® Bisulfite Conversion Kit permits complete conversion of unmethylated cytosine to uracil in reactions that minimize DNA damage and maximize unbiased recovery of DNA. Step 2: Primer Design PCR primer design for MS-HRM is critical— the free online tool from Applied Biosystems, Methyl Primer Express® Software v1.0, was developed specifically for primer design in methylation studies (www.appliedbiosystems .com/methylprimerexpress). We recommend designing several sets of PCR primers for each genomic DNA region of interest, testing them for specificity and the ability to discriminate methylated from unmethylated DNA, and selecting the best-performing assay(s) for analysis of experimental samples. There are three main criteria to consider in designing methylSEQr® Bisulfite Conversion Kit Methyl Primer Express® Software MeltDoctorTM HRM reagents ViiATM 7 Real-Time PCR System 7900HT Fast Real-Time PCR System 7500 Fast Real-Time PCR System StepOnePlusTM Real-Time PCR System StepOneTM Real-Time PCR System ViiATM 7 Software HRM Module High Resolution Melt Software v2.0 High Resolution Melt Software v3.0 Veriti® 96-Well Fast Thermal Cycler 3130 Genetic Analyzer BigDye® Terminator v1.1 Cycle Sequencing Kit BigDye XTerminator® Purification Kit 1 Bisulfite Conversion of DNA 2 Primer Design 3 High-Resolution Melting Optimization 5 Sequencing HRM Product 4 Establishing MS-HRM Standard Curves and Sample Analysis Figure 1. Applied Biosystems Workflow for Methylation-Sensitive High-Resolution Melting (MS-HRM) Analysis Followed by DNA Sequencing. APPLICATION NOTE Methylation Analysis Using Methylation‐Sensitive HRM and DNA Sequencing