Biomedical Engineering Reference
In-Depth Information
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Figure 3.1
Agarose gel electrophoresis of quality control PCR amplification of DNA
prepared from FFPE tissues. Lanes 1-9 contain DNA samples prepared from different FFPE
tumor samples. Lanes 1-8 exemplify 'acceptable' DNA and lane 9 'unacceptable' DNA. The
left lane contains a molecular weight marker (Smartladder; Eurogentec). All the samples
are analyzed on a 2 % (w/v) agarose gel in Tris-acetate-EDTA (TAE) buffer.
3.2.5 Loss of heterozygosity
LOH of chromosomal regions is a common characteristic of cancer [41], and their detection
provides insight into the genomic regions that might harbor potential tumor-suppressor
genes. LOH was originally analyzed using simple sequence-length polymorphisms and is
found through the genotyping of tumor and normal DNA from the same patient. LOH
is identified as the regions where the heterozygous markers in the normal DNA become
homozygous in the tumor. LOH detected at the genotype level can be a result of allelic
imbalances (AIs) that can be caused by either physical loss or gain of alleles. In addition,
mitotic recombination leads to LOH. With the advent of the SNP arrays, high-throughput
analysis of LOH in cancer has become possible; this was first described in a study on
small-cell lung carcinomas [42]. Moreover, the identification of the haplotypes that are
lost in a tumor could be deployed in the study of hereditary cancer. An explosion of
studies on LOH in various cancers has been observed. The methods to identify and visualize
LOH are being developed and are improving. Since paired normal samples for tumors and
cell lines are not always available, the efforts to infer LOH from tumor samples only are
noteworthy [43, 44]. The application of high-throughput SNP arrays for genotyping and
LOH analysis in FFPE samples is a major advance and opens up the tissue archives for
these studies.
We do not present the Illumina protocol for genotyping and LOH analysis, using the
Illumina Goldengate assay and Sentrix arrays, as it is a commercial platform (the most
recent version of the protocol can be obtained through www.illumina.com). Alternatively,
we discuss data management, and the analysis of genotypes and LOH, including the use of
a chromosome visualization tool [45] in Spotfire DecisionSite (see Protocols 3.5 and 3.6).
