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Molecular cytogenetics is the study of chromosome structure involving a combination of molecular biology and cytogenetics. Molecular cytogenetics provides a close look at the chromosomes in a cell and can be used to reveal structural changes between normal and mutated cells. Changes may exist at the chromosomal level, such as chromosome number, or at a smaller scale. An example of a smaller scale change is duplication or translocation of a chromosomal segment. Molecular cytogenetics has many applications in medicine and research, and is a useful diagnostic tool for genetic disorders and cancer.

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. Signals from the fluorescent tags can be seen with microscopy, and mutations can be seen by comparing these signals to healthy cells. FISH can be applied directly to chromosomes in actively dividing or non-dividing cells to observe changes in genetic composition at a molecular level.

FISH chromosome in-situ suppression hybridization allows the study cytogenetics in pre- and postnatal samples. This technique labels all individual chromosomes at every stage of cell division to display structural and numerical abnormalities that may arise throughout the cycle.

Alternatively, an indirect approach can be taken in which the entire genome is be assessed for copy number changes. This technique is referred to as virtual karyotyping. Virtual karyotypes are generated from arrays that hold thousands to millions of probes, and computational tools are used to analyze and assemble the genome in silico.

Comparative Genomic Hybridization (CGH)
Comparative genomic hybridization (CGH) 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. 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.

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.

Uses
Cancer cells often accumulate complex chromosomal structural changes such as loss, duplication, inversion or movement of a segment. By using FISH, any changes to a chromosome will be made visible through discrepancies between fluorescent-labelled cancer chromosomes and healthy chromosomes. The findings of these cytogenetic experiments can shed light on the genetic causes for the cancer and locate potential therapeutic targets.

Molecular cytogenetics can also be used as a diagnostic tool for congenital syndromes in which the underlying genetic causes of the disease are unknown. Analysis of a patient's chromosome structure can reveal causative changes. New molecular biology methods developed in the past two decades such as next generation sequencing and RNA-seq have largely replaced molecular cytogenetics in diagnostics, but recently the use of derivatives of FISH such as multicolour FISH and multicolour banding (mBAND) has been growing in medical applications.

Molecular Cytogenetics Cancer Projects
The Cancer Genome Characterization Initiative (CGCI) is a current research project utilizing molecular cytogenetics to uncover mutations involved in rare cancers. The project utilizes, by advanced genome sequencing, exomes and transcriptomes, which often play a role in the development of cancer. The CGCI has uncovered several previously unknown genetic alterations, such as in medulloblastoma and B-cell non-Hodgkin's lymphoma. The CGCI is currently putting efforts into identifying genomic alternations in HIV and in Burkitt's Lymphoma.

Some of the high-throughput sequencing techniques that the CGCI employed include whole genome sequencing, transcriptome sequencing, ChIP-sequencing, and Illumina Infinum MethylationEPIC BeadCHIP.