Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF

Abstract

Here, we introduce a method for the fast and accurate analysis of DNA methylation based on bisulfite-treated DNA. The target region is PCR amplified using a T7 RNA polymerase promoter-tagged primer. A subsequent in vitro transcription leads to a transcript which contains guanosine residues only at sites that contained methylated cytosines before bisulfite treatment. In a single tube reaction using guanosine-specific cleavage by RNase T1, a specific pattern of RNA fragments is formed. This pattern directly represents the methylation state of the sample DNA and is analyzed using matrix-assisted laser desorption ionization time-of-flight technology. This method was successfully applied to the analysis of artificially methylated and unmethylated DNA, mixtures thereof and colon DNA samples. The applicability for the analysis of both PCR products and cloned PCR products is demonstrated. The observed methylation patterns were confirmed by bisulfite sequencing.

Figure 1

CpG methylation pattern analysis using RNase T1 cleavage and MALDI-TOF: (I) deamination of unmethylated cytosines to uracils by bisulfite treatment, (II) PCR amplification with the reverse primer containing a T7 promoter site and the forward primer carrying a control tag (yellow), (III) transcription to RNA containing G-sites at originally methylated cytosines (^me5C) and (IV) G-specific cleavage with RNase T1.

Figure 2

(I) Multiple alignment of the virtual sequences of the investigated T7 transcripts derived from the bisulfite sequence traces of: PCR product from methylated, bisulfite-treated DNA (A), PCR product from unmethylated, bisulfite-treated DNA (B), PCR product from plasmid pEPI2383 DNA (C) and plasmid pEPI2383 without further PCR (D). Guanosines derived from originally methylated cytosines are highlighted in red, from non-converted cytosines in blue and from the residual vector, T7 Promoter and control tag in yellow. Gene-specific priming sites are marked in italic. (II) Fragmentation (sequences, their related m/z values and positions of the represented CpG) of the T7 transcript A (in I). Fragments from the T7 domain of the primer are tagged with P1–P4, gene-specific fragments are labeled with numbers (1–14) and fragments from the attached control tag are marked with C1–C2. Fragments size >11000 Da and <1000 Da could not be detected. These are highlighted in italic. Mass accuracy for the mass range 2000–10000 m/z is ± 3. (III) MALDI TOF spectra of the base-specific cleaved T7 transcripts A, B, C and D (in I).

Figure 3

Analysis of CpG methylation in 10 clones (A–J) each derived from two bisulfite-converted colon tumor DNA samples (T1 and T2) and two normal colon DNA samples (N1 and N2), respectively, by RNA cleavage and MALDI-TOF (left) and sequencing (right). CpG methylated (black circle); CpG unmethylated (white circle); no fragment indicative of CpG methylation observed or ambiguous sequence (gray circle); CpG not accessible for analysis (cross).

Figure 4

Direct analysis of two bisulfite-converted colon tumor DNA samples (T1 and T2) and two normal colon DNA samples (N1 and N2), respectively, by PCR, in vitro transcription, RNAse T1 cleavage and MALDI-TOF in comparison with DNA mixtures with defined methylation states. (top, mass spectrum of samples T1, T2, N1, N2, respectively; bottom, spectra of DNA mixtures with different, defined methylation states and signal intensity of control fragment C1 set as maximum, fragment numbers given for 100%).