Figure 1: mTAG labelling-based enrichment and analysis of unmethylated CpG sites in the genome.
From: DNA unmethylome profiling by covalent capture of CpG sites
(a) Flow diagram of the analytical procedure. gDNA is randomly sheared to short fragments (Step 1) and treated with an engineered SssI DNA methyltransferase (eM.SssI) and a cofactor analogue (Ado-6-amine or azide) to attach reactive groups to unmodified CpG sites (Step 2). The derivatized target sites are biotin-tagged using N-hydroxysuccinimidyl ester (Biotin-SS-NHS) (Step 3) and labelled fragments are selectively captured on streptavidin-coated magnetic beads (Step 4). Bound DNA fragments are recovered by cleavage of a disulphide bond in the biotin linker with DTT (Step 5). The enriched fragments are ligated to adaptors and PCR-amplified (Step 6) for microarray analysis or DNA sequencing (Step 7). (b) Covalent transformations during derivatization, biotin tagging and linker cleavage (Steps 2, 3 and 5) using amine-NHS (upper) or azide-DBCO (lower) conjugation chemistries (one of the two triazol regioisomers formed in Step 3 is shown); (c) Affinity capture of DNA fragments containing unmodified CpG sites. Reference DNA fragments, containing two or no CpG sites (2-CG and 0-CG, respectively) was each combined with 300 ng of sonicated human gDNA and processed as described (Steps 1–4) using the amine or azide conjugation chemistries as indicated. The efficiency of CpG capture is assessed by on-beads qPCR analysis of the reference DNA fragments. Error bars defined as ±s.d. from duplicate experiments.
