Biomedical Engineering Reference
In-Depth Information
high-density SNP arrays can interrogate more than a million SNPs, these arrays cannot
(yet) be used reliably with DNA from FFPE tissue. This is due to the fragmented nature
of the DNA isolated from FFPE tissue. For this purpose, current arrays are restricted to
6000
10 000 features.
Different methodologies and types of commercially available SNP arrays exist. These
consist of either locus-specific arrays of oligonucleotides (Genechips) or of arrays with
universal capture oligonucleotide on beads that are randomly assembled on arrays and
subsequently decoded (Beadarrays). Genechips can detect up to 250 000 SNPs on a
single chip. For each SNP, a set of locus-specific oligonucleotides is synthesized on the
array. The sample is prepared according to a whole-genome sampling assay [20]. After
restriction enzyme digestion of high molecular weight genomic DNA and ligation of
a common adaptor, the DNA is amplified in a single-primer PCR and hybridized to a
locus-specific array [21]. For Infinium arrays, genomic DNA is whole-genome amplified,
subsequently fragmented, and denatured DNA is hybridized to a locus-specific array. An
allele-specific primer extension assay on the array is followed by staining and scanning
of the arrays using standard immunohistochemical detection methods. Currently these
arrays can detect over a million SNPs on a single array [22]. Goldengate genotyping
makes use of a multiplex mixture of probes for 96, 384, 768 or 1536 SNPs per array
[23]. For each SNP, a combination of allele-specific and locus-specific primers is annealed
to the SNP locus, the primers are tailed with common forward and reverse primers
and a complementary universal capture probe to the locus-specific primer. Subsequent
allele-specific primer-extension, followed by ligation, generates an allele-specific artificial
PCR template. This template is then PCR-amplified and labeled. After hybridization to
an array of universal-capture probes, the array is scanned in two colors, representing
the two alleles of an SNP. Molecular-inversion probe (MIP) genotyping utilizes a pool
of circularizable locus-specific probes with a multiplexing degree of over 10 000 SNPs
per array. The 5' and 3' ends of each probe anneal upstream and downstream of the
SNP. The 1 bp gap is filled; subsequent ligation seals the nick and generates a circular
probe. Restriction digestion then releases the circularized probe and the resultant product
is PCR-amplified using common primers [24]. The four nucleotide reactions are labeled
in different colors and pooled. Subsequently, the pool is hybridized to an array of
universal-capture probes and the four colors are read out in a scanner. Whereas the
high-density Genechip and Infinium arrays are designed for use with high-quality genomic
DNA, both the Goldengate and the MIP assay can be used to detect LOH and copy number
changes in FFPE tissue [25, 26].
SNP aCGH collects both intensity and allelic information from a sample. To extract
profiles of LOH and copy number abnormalities, different methods and algorithms have
been reported [13,27-30]. For the interpretation of LOH and copy number abnormalities,
specifically for the Goldengate assay, a limited or no method was available. Therefore,
to interpret the Beadarray data an R-package BeadArraySNP was developed. The package
deals with the normalization of the allele-specific signal intensities and the representation
of the copy number and LOH profiles [25].
Here we describe the use of the GoldenGate assay and Beadarrays to generate
high-resolution copy number profiles and LOH using DNA isolated from FFPE tissue
(Protocol 1.8). We do not present Illumina protocols, since this is a commercial platform.
The most recent version of the protocol (user card) can be obtained through www
.illumina.com.
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