3.2 Microarray Technology for Sequencing
A DNA microarray is a collection of DNA segments of known sequence attached in
a known pattern to a solid surface. The DNA to be sequenced is fragmented and
modified with fluorescent tags, which are incorporated through PCR. These DNA
fragments are hybridized to specific sites on the array and can be fluorescently
imaged [68]. Differences in hybridization can be quantified using the fluorescence
intensity [68]. Microarrays are often coupled to antibody enrichment or used with
cDNA to explore RNA abundance under different treatment conditions. The microarray methodologies require small sample input and are easily replicated for multiple
samples.
Commercial arrays have been developed to explore methylation status of human
samples using bisulfite-treated DNA and offer around 850,000 probes for hybridization [69–73]. In this way, a DNA microarray enables high-throughput screening of
many genomic regions [72, 73]. In 2002, Gitan et al. developed the first methylationspecific oligonucleotide (MSO) microarray for DNA methylation analysis [69]. In
this technique, DNA samples are treated with bisulfite to convert cytosine to uracil,
whereas 5mC remains unchanged (Fig. 7a). Specific CpG-containing regions of the
genome are then amplified by PCR and labeled using fluorescent dye-conjugated
primers and hybridized to oligonucleotide probes attached to a glass surface. The
oligonucleotide probes are specifically designed to discriminate between bisulfiteconverted and bisulfite-unconverted sequences containing known CpG sites. The
quantitative differences in hybridization, which can be assessed by the fluorescence
intensity, indicate the methylation status of a particular genomic locus. This first use
of a MSO microarray successfully mapped methylated CpG sites within the CpG
island of the estrogen receptor gene in cultured breast cancer cells and clinical
samples [69].
Further developments in the MSO microarray methodologies have led to a
creation of gene-specific, multiplex systems that can be used to simultaneously
examine methylation across multiple CpG sites of numerous genes. This method
provides both qualitative and quantitative DNA methylation data [70]. Due to these
advantages, the MSO microarray technology was commercialized and is now widely
used to study cytosine methylation along the genome.
A variety of commercially available methylation microarrays are available from
companies such as Illumina, Affymetrix, Agilent, and Roche NimbleGen. One such
commercially available microarray is the Infinium Human Methylation
450 BeadChip (Infinium Methylation 450 K) by Illumina. The Infinium 450 K
array makes it possible to assess the methylation status of more than 480,000
cytosines distributed over the entire human genome [71]. This microarray utilizes
a similar technique as the MSO microarray developed by Gitan et al. in 2002 and can
determine the methylation status of 96% of CpG islands and 99% of known human
genes using ~500 ng DNA [49]. The CpG sites being interrogated include promoter
Experimental Methodologies for Detection and Mapping of Epigenetic DNA Marks
501
A DNA microarray is a collection of DNA segments of known sequence attached in
a known pattern to a solid surface. The DNA to be sequenced is fragmented and
modified with fluorescent tags, which are incorporated through PCR. These DNA
fragments are hybridized to specific sites on the array and can be fluorescently
imaged [68]. Differences in hybridization can be quantified using the fluorescence
intensity [68]. Microarrays are often coupled to antibody enrichment or used with
cDNA to explore RNA abundance under different treatment conditions. The microarray methodologies require small sample input and are easily replicated for multiple
samples.
Commercial arrays have been developed to explore methylation status of human
samples using bisulfite-treated DNA and offer around 850,000 probes for hybridization [69–73]. In this way, a DNA microarray enables high-throughput screening of
many genomic regions [72, 73]. In 2002, Gitan et al. developed the first methylationspecific oligonucleotide (MSO) microarray for DNA methylation analysis [69]. In
this technique, DNA samples are treated with bisulfite to convert cytosine to uracil,
whereas 5mC remains unchanged (Fig. 7a). Specific CpG-containing regions of the
genome are then amplified by PCR and labeled using fluorescent dye-conjugated
primers and hybridized to oligonucleotide probes attached to a glass surface. The
oligonucleotide probes are specifically designed to discriminate between bisulfiteconverted and bisulfite-unconverted sequences containing known CpG sites. The
quantitative differences in hybridization, which can be assessed by the fluorescence
intensity, indicate the methylation status of a particular genomic locus. This first use
of a MSO microarray successfully mapped methylated CpG sites within the CpG
island of the estrogen receptor gene in cultured breast cancer cells and clinical
samples [69].
Further developments in the MSO microarray methodologies have led to a
creation of gene-specific, multiplex systems that can be used to simultaneously
examine methylation across multiple CpG sites of numerous genes. This method
provides both qualitative and quantitative DNA methylation data [70]. Due to these
advantages, the MSO microarray technology was commercialized and is now widely
used to study cytosine methylation along the genome.
A variety of commercially available methylation microarrays are available from
companies such as Illumina, Affymetrix, Agilent, and Roche NimbleGen. One such
commercially available microarray is the Infinium Human Methylation
450 BeadChip (Infinium Methylation 450 K) by Illumina. The Infinium 450 K
array makes it possible to assess the methylation status of more than 480,000
cytosines distributed over the entire human genome [71]. This microarray utilizes
a similar technique as the MSO microarray developed by Gitan et al. in 2002 and can
determine the methylation status of 96% of CpG islands and 99% of known human
genes using ~500 ng DNA [49]. The CpG sites being interrogated include promoter
Experimental Methodologies for Detection and Mapping of Epigenetic DNA Marks
501