Exact location of a genetic loci is identified by Fluorescent in-situ hybridization (FISH).Fluorescent in-situ hybridization:FISH, which utilizes fluorescent rather than radioactiveiy labeled probes, is a very sensitive technique for exact location of a genetic lociQ.This often places the gene at a location on a given band or region of the chromosomeQ.Polymerase chain reaction:PCR is a cell free, test tube method used for amplifying a target sequence of DNAQ.Much faster and more sensitive than cell based cloningQPCR is used to amplify specific regions of DNA strandQ (target DNA)Chromosome painting:A technique for visualizing CHROMOSOME ABERRATIONS using fluorescently labeled DNA probes, which are hybridized to chromosomal DNA.Multiple fluorochromes may be attached to the probes.Upon hybridization, this produces a multicolored, or painted, effect with a unique color at each site of hybridizationQ.This technique may also be used to identify cross-species homology by labeling probes from one species for hybridization with chromosomes from another species.Comparative genomic hybridization:Comparative genomic hybridization differentiate the chromosome of normal and cancer cellsQ.Comparative genomic hybridization is used to study cryptic chromosomal imbalances in patients with mental retardation and multiple congenital anomalies, as well as in prenatal diagnosis and to detect microdeletions and microduplications in cancer and in previously unidentified genomic disordersQ.FISH (Fluorescent In-Situ Hybridization)The majority of FISH applications involve hybridization of one or two probes of interest as an adjunctive procedure to conventional chromosomal banding techniques.In this regard. FISH can be utilized to identify specific chromosomes, characterize de novo duplications or deletions, and clarify subtle chromosomal rearrangementsQ.Its greatest utilization in constitutional analysis, however, is in the detection of microdeletionsQ.In cancer cytogenetics, it is used extensively in the analysis of structural rearrangements.Though conventional cytogenetic studies ean detect some microdeietions, initial detection and/or confirmation with FISH is essential.In addition to metaphase FISH, cells can be analyzed at a variety of stages. Interphase analysis can be used to make a rapid diagnosis in instances when metaphase chromosome preparations are not yet available (e.g., amniotic fluid interphase analysts).One specialized type of interphase analysts involves the application of FISH to paraffin-embedded sections, thereby preserving the architecture of the tissue.The use of interphase FISH has increased recently, especially for analyses of amniocentesis samplesQ.These studies are performed on uncultured amniotic fluid, typically using DNA probes specific for the chromosomes most commonly identified in trisomies (chromosomes 13, 18, 21, and the X and Y).These studies can be performed rapidly (24-48 hours) and will ascertain about 60% of the abnormalities detected prenatally.Interphase analysts is routinely utilized is cancer cytogeneticsQ.Many site-specific translocations are associated with specific types of malignanciesQ.For example, there are probes available for both the Abelson (Abl) oncogene and breakpoint cluster region (ber) involved in chronic myelogenous leukemia (CML); these probes are labeled in red and green, respectively; the fusion of these genes in CML combines the fluorescent colors and appears as a yellow hybridization signal.Enhanced techniques for specific types of analysis:Multicolor FISH techniquesReverse paintingFiber FISHComparative genomic hybridizationSpectral karyotyping (SKY) and multicolor FISH (m-FISH) techniquesSKY and m-FISH techniques use combinatorially labeled probes that create a unique color for individual chromosomes.Useful in the identification of unknown chromosome material (such as accessory marker chromosomes containing additional material) but has been most commonly used with the complex rearrangements seen in cancer specimens.Fiber FISHFiber FISH is a technique in which chromosomes are mechanically stretched, using a variety of different methods.It provides a higher resolution of analysis than conventional FISH.FISH comparative genomic hybridization (CGH)FISH comparative genomic hybridization (CGH) is a method that can be used only when DNA is available from a specimen of interestQ.The entire DNA specimen from the sample of interest is labeled in one color (e.g.. green), and the normal control DNA specimen is indicated by another color (e.g.. red). These are mixed in equal amounts and hybridized to normal metaphase chromosomes. The red-to-green ratio is analyzed by a computer program that determines where the DNA of interest may have gains or losses of material.The major advance for examining human chromosomes is an extension of the FISH CGH technologies.Used to study cryptic chromosomal imbalances in patients with mental retardation and multiple congenital anomalies, as well as in prenatal diagnosisQ.Used to detect microdeletions and microduplications in cancer and in previously unidentified genomic disordersQ">