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产品信息
Genomic DNA from many organisms has modified nucleotides. In the mammalian genome, the modified base is predominately 5-methylcytosine (5-mC), which is involved in gene expression regulation, including selective inactivation of one X chromosome in females of mammalian species (1,2). Symmetrical CpG DNA methylation is heritable, but also reversible. This change of methylation status results in an enormous number of combinations of epigenetic states that can regulate gene expression. In mammalian cells, DNA methylation mainly occurs in CpG dinucleotides and is carried out by two methyltransferase enzymatic activities, namely, maintenance methylation and de novo methylation. The maintenance methyltransferase, DNMT1, is involved in the DNA methylation after every cellular DNA replication cycle. DNMT3a and DNMT3b are the de novo methyltransferases that are active in early development.
For optimal results, 50 ng–2 μg DNA is recommended.
Kit Components: Each EpiMark Bisulfite Conversion Kit contains sufficient reagents to perform the bisulfite conversion reaction of 48 samples. Store at room temperature (15–20°C). However, for long-term storage (longer than 4 weeks) Desulphonation Reaction Buffer concentrate should be stored at 2–8°C. Prepared Bisulfite Mix can be stored at -20°C for up to six months.
产品类别:
Discontinued (<3 years)
特性和用法
需要但不提供的材料
Custom design primers EpiMark Hot Start Taq DNA Polymerase (NEB #M0490) or other hot start DNA polymerase specific for bisulfite converted DNA. Heat block or water bath (requiring temperatures of 65°C and 92°C) 96–100% molecular biology grade ethanol PCR Thermal Cycler 0.2 ml strip tubes and caps 1.5 ml reaction tubes Nuclease-free Water Pipettors and pipette tips (for minimization of cross contamination, use aerosol barrier tips)
方法概述
方法概述
Many tools can be used to determine the methylaton status of DNA, including antibodies, methyl binding proteins, methylation-dependent restriction enzymes, and most recently next generation sequencing. However, the most commonly used technique to date is sodium bisulfite conversion, the “gold standard” for methylation analysis (3). Incubation of the target DNA with sodium bisulfite results in conversion of all unmodified cytosines to uracils leaving the modified bases (5-mC or 5-hmC) intact. (see Figure 1). The most critical step in methylation analysis using bisulfite conversion is the complete conversion of unmodified cytosines. This is achieved by alternating cycles of thermal denaturation with incubation reactions. The protocol optimized for this kit is simple and gives consistent results.
Figure 1. Overview of Bisulfite Conversion ReactionMethylated cytosines are protected and remain unchanged, while unmethylated cytosines are deaminated to uracil after treatment with sodium bisulfite.Figure 2. Performance evaluation of the EpiMark Bisulfite Conversion Kit.One μg of genomic DNA was bisulfite treated using the Epimark Bisulfite Conversion Kit, and 2 μl of eluted DNA was analyzed by end-point PCR. 388, 544, and 731 bp amplicons were amplified with primer pairs for bisulfite converted DNA (lanes 1, 3, and 5), or with primer pairs for unconverted DNA (lanes 2, 4, and 6). Epimark Hot Start Taq DNA Polymerase was used for amplification. Lanes 2, 4, and 6 clearly show no amplification product, indicating complete DNA conversion. Marker M is the 2-Log DNA Ladder (NEB #N3200).Figure 3. Conversion of a methylated DNA fragment.50 nanograms of methylated plasmid DNA was bisulfite converted and a 146 bp DNA fragment was amplified using Epimark Hot Start Taq DNA Polymerase (NEB #M0490). Amplicons were cloned, and individual clones were sequenced to determine the methylation status. Sequence aligment of 10 clones is shown to demonstrate conversion of unmethylated cytosine to thymine, while the unconverted methylated cytosine remains intact. The known methylated cytosines at the CpG site shown in red, converted cytosines marked as T, and rest of the sequence is shown as a dashed line.
