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.
- Chromosomal Banding - Other Banding
- Chromosomal Banding - Chromosome Structure
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