Molecular cytogenetics

Common techniques

Fluorescence in situ hybridization (FISH)

Fluorescence In Situ Hybridization maps out single copy or repetitive DNA sequences through localization labeling of specific nucleic acids. The technique utilizes different DNA probes labeled with fluorescent tags that bind to one or more specific regions of the genome. It labels all individual chromosomes at every stage of cell division to display structural and numerical abnormalities that may arise throughout the cycle. This is done with a probe that can be locus specific, centromeric, telomeric, and whole-chromosomal. This technique is typically performed on interphase cells and paraffin block tissues. FISH maps out single copy or repetitive DNA sequences through localization labeling of specific nucleic acids. The technique utilizes different DNA probes labeled with fluorescent tags that bind to one or more specific regions of the genome. Signals from the fluorescent tags can be seen with microscopy, and mutations can be seen by comparing these signals to healthy cells. For this to work, DNA must be denatured using heat or chemicals to break the hydrogen bonds; this allows hybridization to occur once two samples are mixed. The fluorescent probes create new hydrogen bonds, thus repairing DNA with their complementary bases, which can be detected through microscopy. FISH allows one to visualize different parts of the chromosome at different stages of the cell cycle. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from microarrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico.

Comparative genomic hybridization (CGH)

Comparative genomic hybridization (CGH), derived from FISH, is used to compare variations in copy number between a biological sample and a reference. CGH was originally developed to observe chromosomal aberrations in tumour cells. This method uses two genomes, a sample and a control, which are labeled fluorescently to distinguish them. In CGH, DNA is isolated from a tumour sample and biotin is attached. Another labelling protein, digoxigenin, is attached to the reference DNA sample. The labelled DNA samples are co-hybridized to probes during cell division, which is the most informative time for observing copy number variation. CGH uses creates a map that shows the relative abundance of DNA and chromosome number. By comparing the fluorescence in a sample compared to a reference, CGH can point to gains or losses of chromosomal regions. CGH differs from FISH because it does not require a specific target or previous knowledge of the genetic region being analyzed. CGH can also scan an entire genome relatively quickly for various chromosome imbalances, and this is helpful in patients with underlying genetic issues and when an official diagnosis is not known. This often occurs with hematological cancers.

Array comparative genomic hybridization (aCGH)

Array comparative genomic hybridization (aCGH) allows CGH to be performed without cell culture and isolation. Instead, it is performed on glass slides containing small DNA fragments. Removing the cell culture and isolation step dramatically simplifies and expedites the process. Using similar principles to CGH, the sample DNA is isolated and fluorescently labelled, then co-hybridized to single stranded probes to generate signals. Thousands of these signals can be detected for at once, and this process is referred to as parallel screening. Fluorescence ratios between the sample and reference signals are measured, representing the average difference between the amount of each. This will show if there is more or less sample DNA than is expected by reference.