Method for methylation analysis
A technique for methylated and unmethylated cytosine, used in the field of assessment of DNA methylation
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example 1
[1233] Adverse effects of bisulfite treatment on PCR-amplified DNA.
[1234] To determine how much bisulfite treatment affected PCR amplification, droplet digital PCR (ddPCR) was used to compare the quantification of amplicons before and after bisulfite treatment.
[1235] Concentrations of fully methylated human genomic DNA (Millipore) were estimated by measuring absorbance at 260 nm following standard practice. The expected equivalent DNA copy number was calculated and a portion of the DNA was treated with sodium bisulfite. The actual amount of PCR-amplified DNA copies before and after bisulfite treatment was determined by ddPCR using the ACTB assay. Such as figure 1As shown, the ddPCR ACTB assay used to estimate DNA copies before bisulfite treatment detects and amplifies both complementary strands in the region of interest [SEQ ID 67-71], whereas after bisulfite treatment for Assays that estimate DNA copies detect and amplify only one strand of DNA [SEQ ID 72-76]. Ther...
example 2
[1240] Targets the double strand of bisulfite-converted DNA
[1241] Fully methylated human genomic DNA (2000 pg / well; Millipore) was used as template and bisulfite conversion was performed on a QIAcube HT (Qiagen) using a custom-made Epitect Rapid DNA Bisulfite Kit (Qiagen). qPCR assays targeting regions on one or both strands of BCAT1 [SEQ ID 64-66 and 61-63] and IKZF1 [SEQ ID 11-21 and 22-32] were designed. The PCR reaction consisted of 15 μL of 2x Quantitect mastermix, 3 μL per 200 nM of forward and reverse primers and 3 μL per 100 nM of probe with nuclease-free water, and 12 μL of template DNA (166.7 pg / μL), cycled under the following conditions: (Light Cycler) 480 (Roche), 95°C, 15 minutes; [95°C, 15 seconds; 62°C, 40 seconds] x50; 40°C, 10 seconds.
[1242] figure 2 Amplification results for A) BCAT1 and B) IKZF1 are shown. It can be seen that BCAT1 and IKZF1 generate approximately twice the amount of amplicons when using primers targeting both strands of DNA compar...
example 3
[1244] ddPCR quantification of single- or double-stranded amplification of bisulfite-converted DNA
[1245] Droplet digital PCR (ddPCR) provides absolute quantification by distributing DNA into approximately 20,000 discrete droplets, which are then used as individual reaction vesicles for endpoint PCR amplification. After PCR amplification, the droplets were evaluated for fluorescence as an indicator that at least one DNA molecule was initially contained in that particular droplet. Count the number of fluorescent positive droplets and calculate the DNA copy number in the original sample using a Poisson distribution to model the likelihood that each droplet contains one or more DNA molecules. Because PCR amplification is the end point, the efficiency of the reaction is not critical, and the quantification provided by this technique is a more accurate measure.
[1246] To confirm whether the phenomenon observed in Example 2 is independent of PCR amplification efficiency, the ab...
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