Journal: BMC Molecular Biology
Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
Figure Lengend Snippet: Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length
Article Snippet: Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA).
Techniques: Methylation, Ligation, Polymerase Chain Reaction, Amplification