Method
The process is outlined in Figure 1.
Bisulfite treatment
Approximately 1 ug of genomic DNA is used in bisulfite conversion to convert the unmethylated cytosine into uracil. The product contains unconverted cytosine where they were previously methylated, but cytosine converted to uracil if they were previously unmethylated.
Whole genomic DNA amplification
The bisulfite treated DNA is subjected to whole genome amplification (WGA) via random hexamer priming and Phi29 DNA polymerase, which has a proofreading activity resulting in error rates 100 times lower than the Taq polymerase. The products are then enzymatically fragmented, purified from dNTPs, primers and enzymes, and applied to the chip.
Hybridization and Single-base extension
On the chip, there are two bead types for each CpG site per locus. Each locus tested is differentiated by different bead types, there are over 200,000 bead types available. Each of the bead types are attached to a single stranded 50-mer DNA oligonucleotides that differ in sequence only at the free end; this type of probe is known as an Allele specific oligonucleotide. One of the bead types will correspond to the methylated cytosine locus and the other will correspond to the unmethylated cytosine locus, which has been converted into uracil during bisulfite treatment and later amplified as thymine during whole genome amplification. The bisulfite converted amplified DNA products are denatured into single strands and hybridized to the chip via allele specific annealing to either the methylation specific probe or the non-methylation probe . Hybridization is followed by single base extension with hapten labelled dideoxynucleotides. The ddCTP is labelled with biotin while ddATP, ddUTP and ddGTP are labelled with 2,4-dinitrophenol (DNP)
Fluorescence staining and scanning of chip
After incorporation of these hapten labelled ddNTPs, multi-layered immunohistochemical assays are performed by repeated rounds of staining with a combination of antibodies to differentiate the two types. After staining, the chip is scanned to show the intensities of the unmethylated and methylated bead types. (Figure 2). The raw data are analyzed by the proprietary software, and the fluorescence intensity ratios between the two bead types are calculated. A ratio value of 0 equals to non-methylation of the locus; a ratio of 1 equals to total methylation; a value of 0.5 means that one copy is methylated and the other is not, in the diploid human genome.
Analysis of methylation data
The scanned microarray images of methylation data are further analyzed by the system, which normalizes the raw data to reduce the effects of experimental variation, background and average normalization, and performs standard statistical tests on the results. The data can then be compiled into several types of figures for visualization and analysis. Scatter plots are used to correlate the methylation data; bar plots to visualize relative levels of methylation at each site tested; heat maps to cluster the data to compare the methylation profile at the sites tested. Figure 2 shows the different types of results generated.
Read more about this topic: Illumina Methylation Assay
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