Background

DNA methylation is a conserved chemical modification at the 5’ position in the deoxyribose ring of cytosine that is catalyzed by methyltransferases (Figure 2A and Schultz et al., 2012). In plants, DNA methylation occurs in three sequence contexts: CG, CHG, and CHH, where H represents A, T, or C (Zhang et al., 2018). DNA methylation plays an important role in regulating both genes and transposable elements (TEs) (Schultz et al., 2012; Zhang et al., 2018; Guo et al., 2021). Using a maize MuDR reporter system to study TE silencing, we have demonstrated that DNA methylation at a terminal inverted region (TIR) in one of the MuDR elements, mudrA, is associated with silencing (Woodhouse et al., 2006; Guo et al., 2021).

Bisulfite sequencing involves bisulfite treatment of genomic DNA, resulting in the conversion of unmethylated cytosines into uracil, while methylated cytosines remain unchanged. After treatment, the region of interest is amplified, cloned, and sequenced. Investigating DNA methylation at a gene or a TE requires high-quality genomic DNA, and the conversion of unmethylated cytosines into uracil, while retaining methylated cytosines using sodium bisulfite treatment (Gruntman et al., 2008; Zhang et al., 2018). After this treatment, the region of interest is amplified via polymerase chain reaction (PCR), which is followed by TA-cloning and Sanger sequencing (Foerster, 2010). Because PCR amplification of the converted cytosines will replace uracil with thymine, determining the methylation profile at a region of interest can be achieved by comparing the sequence of the bisulfite-treated DNA with that of untreated DNA sequences (Gruntman et al., 2008). To minimize the noise created by incomplete conversion of unmethylated cytosine to uracil, examining DNA methylation using an internal control gene, and assaying multiple individual colonies of PCR products are needed.

Here, we provide a case study with step-by-step procedures, to examine the pattern of DNA methylation at a silenced MuDR element in maize, which is a well-documented system to validate our protocol. We described a site-specific DNA methylation pipeline, using traditional TA cloning and Sanger sequencing and provide procedures for sample preparation, treatment, cloning work, and data analysis. We highlight the applicability of this traditional DNA methylation pipeline as an inexpensive method for pilot studies, important for decision-making, or for use when an organism’s genome is not fully sequenced. Additionally, this pipeline describes the basis of molecular cloning and epigenetic analysis, and how it can be used as a method for teaching.