DNA Methylation Workflow
To date, a number of methods have been developed to quantify DNA methylation, including high-performance capillary electrophoresis and methylation-sensitive arbitrarily primed PCR (Fraga ., 2000; Gonzalgo , 1997). For gene-specific methylation analysis, bisulfite reaction based methods have become very popular. These entail modifying DNA using sodium bisulfite to convert unmethylated cytosines to uracils. Following bisulfite conversion, the modified DNA template is distinguishable from the original template at methylated cytosines. This results in a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Methylated cytosines are subsequently detected using various proprietary and non-proprietary methods such as methylation specific PCR (MSP) technique or sequencing after PCR amplification with or without cloning. For example, in Figure 1 the methylation profile of the DNA segment is determined by comparing the sequence of the bisulfite-treated DNA to that of the untreated DNA.
Figure 1. DNA sequencing results after bisulfite treatment
Following sequencing, the methylation profile of the DNA segment is determined by comparing the sequence of the bisulfite-treated DNA to that of the untreated DNA.
Genomic DNA Isolation
The PureLink™ Genomic DNA Mini Kit is based on the selective binding of DNA to silica-based membrane in the presence of chaotropic salts. The lysate is prepared from a variety of starting materials such as tissues, cells, or blood. The cells or tissues are digested with Proteinase K at 55°C using an optimized digestion buffer formulation that aids in protein denaturation and enhances Proteinase K activity. Residual RNA is removed by RNase digestion prior to sample binding to the silica membrane. The lysate is mixed with ethanol and PureLink™ Genomic Binding Buffer that allows high DNA binding to the silica-based membrane in the column. Impurities are removed by thorough washing with Wash Buffers 1 and 2. The genomic DNA is then eluted in low salt Elution Buffer.
Genomic DNA undergoes bisulfite treatment with the MethylCode™ Bisulfite Conversion Kit. Starting with 500 pg to 2 μg per treatment (200–500 ng optimal), the MethylCode™ Bisulfite Conversion Kit integrates the DNA denaturation and bisulfite conversion processes into one step, by using temperature denaturation to replace chemical denaturation with sodium hydroxide. Following bisulfite conversion, the DNA is recovered using a streamlined in-column desulphonation method.
The bisulfite converted DNA is used as template in a PCR reaction to amplify a portion on the gene using Platinum™ Taq Supermix or AccuPrime™ Taq Supermix, bisulfite conversion specific primers, bisulfite converted gDNA, in two rounds of amplification. For PCR amplification of the bisulfite-converted DNA, best results are achieved using primers that are 24–26 bases in length. For human and mouse DNAs, all unmethylated C’s (i.e., non-CpG) will be converted into uracils during the bisulfite treatment. Therefore, these C’s should be treated as T’s for primer design purposes. For example, for the sequence 5´- AACCTTACAGGCAC-3´, the corresponding primer should be 5´-AATTTTATAGGTAT-3´.
Cloning for Sequencing
PCR product is cloned into a TOPO TA sequencing vector. The TOPO TA Cloning® Kits for Sequencing provide a highly efficient, 5 minute, one-step cloning strategy for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector for sequencing. No ligase, post-PCR procedures, or PCR primers containing specific sequences are required. Finally, the sequences are analyzed to show a differential methylation pattern of the gene between the cell-lines.
Isolating Plasmid DNA
The PureLink™ Quick Plasmid Miniprep Kit isolates high quality plasmid DNA (up to 30 μµg) from E. coli cells in 30-45 minutes. Cells are lysed using an alkaline/SDS procedure. The lysate is then applied to a silica membrane column that selectively binds plasmid DNA. Contaminants are removed with Wash Buffers. The plasmid DNA is eluted in TE Buffer and is suitable for all routine downstream applications. The PureLink™ Quick Plasmid Miniprep Kit can be used with a centrifuge or a vacuum manifold.