Page 1340 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPTEr 96 Molecular Methods 1301

Label test DNA with Cy3 Label standard control DNA gDNA
with Cy5

WGA

Fragment and
Denature

Scan
Anneal

A G * TA * CG

A B
Hybridize
C Identical probes per bead type Single base extension

Scan

Cy3/Cy5 ratio >1 Cy3/Cy5 ratio <1
A Duplication Deletion
Restriction digest
PM-Sense A
MM-Sense A
Xbal Xbal Xbal gDNA
PM-Antisense A
MM-Antisense A
Adaptor ligation

PM-Sense B
MM-Sense B
PCR End label PM-Antisense B
MM-Antisense B
b
b Hybridize and wash
b
b

B
FIG 96.3 Array-Based DNA Copy Number Analysis. (A) Array comparative genomic hybridization
(aCGH) uses a reference DNA to compare with the test DNA. The labeled DNA is hybridized to
an array of oligonucleotides that are designed to be uniquely complementary to hundreds of
thousands of positions in the genome. The relative intensities of the fluorescence signal from
each DNA probe allow assessment of copy number. Genotyping arrays are also widely used for
copy number testing (B, C). (B) Whole genome sampling and amplification (WGSA). End-labeled
polymerase chain reaction (PCR) products that constitute a sample from the genome are hybridized
to oligo sets on standard chips. The oligos are organized into “probe quartets” consisting of
perfect matches (PMs) and single-base mismatches (MMs) for each allele, A and B. Separate
quartets for the sense and antisense strand of DNA help to ensure specificity, and in the actual
assay these quartets are repeated three to five times on the chip. This type of assay has been
developed to test a standard panel of >2 million DNA features per sample. (C) Single base
extension (SBE; Illumina Infinium II). This assay uses whole genome amplification (WGA) to
increase the molar concentration of target DNA. SBE on arrays of tagged microbeads allows
allele discrimination, and the resulting products are detected by immunostaining (signal amplifica-
tion). This assay has been used for assays of up to 5 million single nucleotide polymorphisms
(SNPs) per sample.