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مساعده حول banding technique

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  • مساعده حول banding technique

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    أريد عمل تقرير عن banding technique


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  • #2
    Chromosomal Banding

    A chromosome banding pattern is comprised of alternating light and dark stripes, or bands, that appear along its length after being stained with a dye. A unique banding pattern is used to identify each chromosome and to diagnose chromosomal aberrations, including chromosome breakage, loss, duplication or inverted segments. In the 1950s, chromosomes from the cell's nucleus were identified with a uniform (unbanded) stain that allowed for the observation of the overall length and primary constriction (centromere) of each chromosome, as well as a secondary constriction in chromosomes 1, 9, 16 and the acrocentrics (chromosomes whose centromeres are near the tips). The staining techniques used to make the bands visible were developed in the late 1960s and early 1970s.
    Chromosome Structure

    To understand what chromosomal bands represent, it is helpful to
    understand the structure of chromosomes. Eukaryotic chromosomes are composed of chromatin, a combination of nuclear DNA and proteins. At metaphase, which is a phase in the cell cycle after the DNA in the nucleus has been replicated, each chromosome contains two identical strands of DNA. (Each strand contains two complementary strands of nucleotides.) The two strands of DNA, or chromatids, are arranged in a double-helix and are held together at a single point, the centromere, or primary constriction point.

    During mitosis , each chromatid becomes condensed approximately ten-thousand fold reaching maximal condensation at metaphase . DNA that was roughly 5 centimeters (2 inches) long is compacted to 5 micrometers. The DNA wraps around proteins called histones, forming complexes called nucleosomes. The nucleosomes twist around each other and assume a loop formation projecting out from the chromosome's protein backbone, or scaffold. The loops weave and condense further to package the DNA into a chromosome. Some of the looped segments of DNA remain close together and condense more than others, forming regions known as domains. These domains are the darkly-stained chromosomal bands that appear when specific stains are applied (such as Giemsa staining; see below).

    Looped domains are also seen in polytene chromosomes, which are found mainly in insects of the order Diptera, including Drosophila, which are fruit flies. Polytene chromosomes are large chromosomes that are formed after DNA undergoes repeated rounds of replication without cell division. A polytene chromosome in a Drosophila salivary gland cell can contain as many as five thousand alternating dark and light bands. The dark bands correspond to the folded and looped DNA, and the lighter bands are composed of less condensed DNA. The DNA in polytene chromosomes becomes less condensed when genes become active, permitting DNA to be transcribed into messenger RNA. This unraveling is observed as "puffing" of the polytene chromosome. The puffing resolves (the DNA condenses again) as the genes become inactive.
    Chromosome Banding Techniques

    Quinacrine mustard, an alkylating agent, was the first chemical to be used for chromosome banding. T. Caspersson and his colleagues, who developed the technique, noticed that bright and dull fluorescent bands appeared after chromosomes stained with quinacrine mustard were viewed under a fluorescence microscope. Quinacrine dihydrochloride was subsequently substituted for quinacrine mustard. The alternating bands of bright and dull fluorescence were called Q bands. Quinacrine-bright bands were composed primarily of DNA that was rich in the bases adenine and thymine, and quinacrine-dull bands were composed of DNA that was rich in the bases guanine and cytosine.

    Other fluorescent dyes have been used to generate chromosomal banding patterns. The combination of the fluorescent dye, DAPI (4,6-Diamidino-2-Phenylindole) with a non-fluorescent counterstain, such as Distamycin A, will also stain DNA that is rich in adenine and thymine. It will particularly highlight regions that are on the Y chromosome, on chromosomes 9 and 16, and on the proximal short arms of the chromosome 15 homologues , or pair.

    Giemsa has become the most commonly used stain in cytogenetic analysis. Staining a metaphase chromosome with a Giemsa stain is referred to as G-banding. Unlike Q-banding, most G-banding techniques require pretreating the chromosomes with either salt or a proteolytic (protein-digesting) enzyme. "GTG banding" refers to the process in which G-banding is preceded by treating chromosomes with trypsin. G-banding preferentially stains the regions of DNA that are rich in adenine and thymine. In general, the bands produced correspond with Q-bright bands. The regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light.

    Standard G-band staining techniques allow between 400 and 600 bands to be seen on metaphase chromosomes. With high-resolution G-banding techniques, as many as two thousand different bands have been catalogued on the twenty-four human chromosomes. Jorge Yunis introduced a technique to synchronize cells so they are held at the same stage in the cell cycle. Cells are synchronized by making them deficient in folate, thereby inhibiting DNA synthesis. By rescuing the cells with thymidine, DNA synthesis is initiated and the timing of the prophase and prometaphase stages of the cell cycle can be predicted. Yunis's technique allows more bands to be resolved, as chromosomes produced from either prophase or prometaphase are less condensed and are thus longer than metaphase chromosomes.
    Other Banding

    R-banding is the reverse pattern of G bands so that G-positive bands are light with R-banding methods, and vice versa. R-banding involves pretreating cells with a hot salt solution that denatures DNA that is rich in adenine and thymine. The chromosomes are then stained with Giemsa. R-banding is helpful for analyzing the structure of chromosome ends, since these areas usually stain light with G-banding.

    C-banding stains areas of heterochromatin, which is tightly packed and repetitive DNA. NOR-staining, where NOR is an abbreviation for "nucleolar organizing region," refers to a silver staining method that identifies genes for ribosomal RNA that were active in a previous cell cycle.

    Fluorescence In Situ Hybridization

    Fluorescence in situ hybridization (FISH) is a molecular cytogenetic
    technique that allows cytogeneticists to analyze chromosome resolution at the DNA or gene level. FISH can be performed on dividing (metaphase) and non-dividing (interphase) cells to identify numerical and structural abnormalities resulting from genetic disorders.

    In FISH, cytogeneticists utilize one or more FISH probes that typically fall into one of the following three categories:

    Repetitive sequences, including alpha satellite DNA, that bind to the centromere of a chromosome;
    DNA segments, representative of the entire chromosome, that will bind to and cover the entire length of a particular chromosome; and
    DNA segments from specific genes or regions on a chromosome that have been previously mapped or identified.
    A probe is "tagged" either directly, by incorporating fluorescent nucleotides, or indirectly, by incorporating nucleotides with attached small molecules, such as biotin, digoxygenin, or dinitrophenyl, to which fluorescent antibodies can later be bound. The probe and the chromosomes (from either the metaphase or interphase cells) that are being analyzed are denatured and allowed to bind or hybridize to one another. If necessary, antibodies with a fluorescent tag are applied to the cells. The cells are then viewed with a fluorescence microscope. The fluorescent signals represent the probe(s) that is bound to the chromosomes.

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