User:GutierrezEmily/Fluorescence in situ hybridization

Preparation and hybridization process – DNA[edit][edit]
Scheme of the principle of the FISH Experiment to localize a gene in the nucleus. First, a probe is constructed. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process. The probe is tagged directly with fluorophores, with targets for antibodies or with biotin. Tagging can be done in various ways, such as nick translation, or Polymerase chain reaction using tagged nucleotides.

Then, an interphase or metaphase chromosome preparation is produced. The chromosomes are firmly attached to a substrate, usually glass. Repetitive DNA sequences must be blocked by adding short fragments of DNA to the sample. The probe is then applied to the chromosome DNA and incubated for approximately 12 hours while hybridizing. Several wash steps remove all unhybridized or partially hybridized probes. The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images

If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of the microscope. Fluorescent signal strength depends on many factors such as probe labeling efficiency, the type of probe, and the type of dye. Fluorescently tagged antibodies or streptavidin are bound to the dye molecule. These secondary components are selected so that they have a strong signal.

'''Above is the original article. below are my edits'''

Preparation and hybridization process – DNA[edit][edit]
Scheme of the principle of the FISH Experiment to localize a gene in the nucleus.

First, a DNA probe is constructed. Typically, DNA probes are derived from bacterial artificial clones (BACs) comprised of a couple hundred kilobase pairs of human genomic DNA. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process . The probe is tagged directly with fluorophores, with targets for antibodies, or with either biotin or digoxigenin. Tagging can be done in various ways, such as nick translation, or polymerase chain reaction using tagged nucleotides.

Then, an interphase or metaphase chromosome preparation is produced. The chromosomes are firmly attached to a substrate, usually glass. Repetitive DNA sequences must be blocked by adding short fragments of DNA to the sample. The probe is then applied to the chromosome DNA and incubated for approximately 12 hours while hybridizing. Several wash steps remove all unhybridized or partially hybridized probes. The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images.

If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of the microscope. Fluorescent signal strength depends on many factors such as probe labeling efficiency, the type of probe, and the type of dye. Fluorescently tagged antibodies or streptavidin are bound to the dye molecule. These secondary components are selected so that they have an amplified, stronger signal.

Locus-specific and "chromosome paint" probes

DNA Probes for FISH can mainly be classified as locus-specific or chromosome painting probes. In the former, probes hybridize to a unique gene or to a specific chromosomal area. They are often used in studies to detect gains, deletions, and rearrangements in genome sequence s. This stands in contrast to chromosome painting probes that consists of a combination of DNA probes and dyes, which together target a complete chromosome in metaphase chromosomes. Many studies utilize this method to detect large segments of sequence such as nuclear chromosomal domains.