the presence of a translocation. Furthermore, a PCR product may be sequenced to look for point mutations.

Fluorescent in situ hybridization (FISH) uses fluorescently labeled DNA probes to bind to specific regions of genomic DNA. Images are then analyzed under a fluorescence microscope. Numerical chromosomal abnormalities may be detected by simply counting the number of signals per cell: more than two indicates the addition of a chromosome, whereas fewer than two indicates a deletion (Plate 10.1). To investigate a potential translocation, two probes are used, one to detect the genomic DNA on each side of the known translocation. If the two probes are consistently approximated, this indicates the presence of a translocation (Plate 10.2). FISH may be performed on interphase cells, so that growth in culture is not a requirement for this type of analysis as it is for conventional cytogenetics. Tests for small deletions or other more subtle abnormalities may be better performed on metaphase cells. FISH requires knowledge of the area to be labeled.

Since they rely on the annealing of a labeled specific DNA probe or primer, Southern hybridization, PCR and FISH are techniques to determine the presence or absence of a known genetic abnormality. Two modern techniques that provide a genome-wide scan for abnormalities and require no prior suspicion of a particular abnormality are comparative genomic hybridization (CGH) and gene expression profiling (GEP). These are beginning to have a clinical impact.

In the original CGH techniques (Figure 10.1), DNA is isolated from the tumor sample and a normal control sample. The DNA in each is labeled with a different fluorescent dye; for example, green for the tumor DNA and red for the normal DNA. These samples are then mixed and hybridized onto slides of metaphase spreads of normal cells. Images of metaphase spreads are then analyzed for the green:red color ratio. Regions of chromosomes that have a high green:red ratio contain a putative area of amplification. Regions that have a low green:red ratio contain a putative deletion. In this way, the entire genome may be examined for abnormalities. Other techniques, generally beyond what is performed in clinical laboratories, are required to determine the critical genes involved in areas of amplification and deletion. Small abnormalities and balanced translocations cannot be observed using this technique. In a variation of the CGH technique, DNA is hybridized to defined arrays of genomic DNA fragments (Figure 10.2). These arrays can cover the genome, and this improves resolution to the size of the DNA fragments within the microarray, currently down to the level of 1 Mb.

GEP is described in more detail in Chapter 24. This technique allows the comprehensive, quantitative examination of the mRNA transcripts of a tumor sample, a group of molecules that has been termed the 'transcriptome'. In this technique, mRNA is purified from a fresh or frozen tumor sample. Formalin-fixed tissue cannot be used. It is important that, when a group of samples is being compared, tissue acquisition, mRNA preparation and all subsequent steps are performed as identically as possible. When possible, steps should be performed on all samples in parallel, with identical reagents, and simultaneously. Techniques exist to amplify very small amounts of mRNA to obtain usable quantities; while

Labeled tumor DNA (rhodamine)

Labeled normal DNA (FITC)

Mix labeled tumor and normal DNAs in equal amounts

Up = tumor DNA amplification Down = loss of tumor DNA

Place DNA on slide

Cover with coverslip

Mix labeled tumor and normal DNAs in equal amounts

Computer analysis

Slide preparation (normal karyotype)

Was this article helpful?

0 0
10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook

Post a comment