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Chromosome Banding

Centromere position and arm ratios can assist in identifying specific pairs of chromosomes, but inevitably several or many pairs of chromosomes appear identical by these criteria. The ability to identify specific chromosomes with certainty was revolutionized by discovery that certain dyes would produce reproducible patterns of bands when used to stain chromosomes. Chromosome banding has since become a standard and indispensible tool for cytogenetic analysis., and several banding techniques have been developed::

Q banding: chromosomes are stained with a fluorescent dye such as quinacrine G banding: produced by staining with Giemsa after digesting the chromosomes with trypsin C banding: chromosomes are treated with acid and base, then stained with Giesma stain

Each of these techniques produces a pattern of dar and light !or fluorescent versus non" fluorescent# bands along the length of the chromosomes. Importantly, each chromosome displays a unique banding pattern, analagous to a "bar code", which allows it to be reliably differentiated from other chromosomes of the same size and centromeric position. $n the above figure, human chromosome pairs %, & and ' are seen with and without G banding.
A chromosome banding pattern is comprised of alternating light and dark stripes, or bands, that appear along its length after being stained with a dye. A unique banding pattern is used to identify each chromosome and to diagnose chromosomal aberrations, including chromosome breakage, loss, duplication or inverted segments. In the 195 s, chromosomes from the cell!s nucleus were identified with a uniform "unbanded# stain that allowed for the observation of the overall length and primary constriction "centromere# of each chromosome, as well as a secondary constriction in chromosomes 1, 9, 1$ and the acrocentrics "chromosomes whose centromeres are near the tips#. %he staining techniques used to make the bands visible were developed in the late 19$ s and early 19& s.

'hromosome (anding %echniques

Quinacrine mustard, an alkylating agent, was the first chemical to be used for chromosome banding. %. 'aspersson 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. )uinacrine dihydrochloride was subsequently substituted for quinacrine mustard. %he alternating bands of bright and dull fluorescence were called ) bands. )uinacrine*bright bands were composed primarily of +,A that was rich in the bases adenine and thymine, and quinacrine*dull bands were composed of +,A that was rich in the bases guanine and cytosine. -ther fluorescent dyes have been used to generate chromosomal banding patterns. %he combination of the fluorescent dye, DAPI ".,$*+iamidino* /*0henylindole# with a non*fluorescent counterstain, such as +istamycin A, will also stain +,A that is rich in adenine and thymine. It will particularly highlight regions that are on the 1 chromosome, on chromosomes 9 and 1$, and on the pro2imal short arms of the chromosome 15 homologues, or pair. 3iemsa has become the most commonly used stain in cytogenetic analysis. 4taining a metaphase chromosome with a 3iemsa stain is referred to as 3*banding. 5nlike )*banding, most 3*banding techniques require pretreating the chromosomes with either salt or a proteolytic "protein*digesting# en6yme. 73%3 banding7 refers to the process in which 3*banding is preceded by treating chromosomes with trypsin. 3*banding preferentially stains the regions of +,A that are rich in adenine and thymine. In general, the bands produced correspond with )*bright bands. %he regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light.

3*banded metaphase from a normal female.

4tandard 3*band staining techniques allow between . and $ bands to be seen on metaphase chromosomes. 8ith high*resolution 3*banding techniques, as many as two thousand different bands have been catalogued on the twenty*four human chromosomes. 9orge 1unis introduced a technique to synchroni6e cells so they are held at the same stage in the cell cycle. 'ells are synchroni6ed by making them deficient in folate, thereby inhibiting +,A synthesis. (y rescuing the cells with thymidine, +,A synthesis is initiated and the timing of the prophase and prometaphase stages of the cell cycle can be predicted. 1unis!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.

-ther (anding
:*banding is the reverse pattern of 3 bands so that 3*positive bands are light with :*banding methods, and vice versa. :*banding involves pretreating cells with a hot salt solution that denatures +,A that is rich in adenine and thymine. %he chromosomes are then stained with 3iemsa. :*banding is helpful for analy6ing the structure of chromosome ends, since these areas usually stain light with 3*banding. '*banding stains areas of heterochromatin, which is tightly packed and repetitive +,A. ,-:*staining, where ,-: is an abbreviation for 7nucleolar organi6ing region,7 refers to a silver staining method that identifies genes for ribosomal :,A that were active in a previous cell cycle.

;luorescence in 4itu <ybridi6ation

;luorescence in situ hybridi6ation ";I4<# is a molecular cytogenetic technique that allows cytogeneticists to analy6e chromosome resolution at the +,A or gene level. ;I4< can be performed on dividing "metaphase# andnon*dividing "interphase# cells to identify numerical and structural abnormalities resulting from genetic disorders. In ;I4<, cytogeneticists utili6e one or more ;I4< probes that typically fall into one of the following three categories= 1. :epetitive sequences, including alpha satellite +,A, that bind to the centromere of a chromosome> /. +,A segments, representative of the entire chromosome, that will bind to and cover the entire length of a particular chromosome> and ?. +,A segments from specific genes or regions on a chromosome that have been previously mapped or identified. A probe is 7tagged7 either directly, by incorporating fluorescent nucleotides, or indirectly, by incorporating nucleotides with attached small

molecules, such as biotin, digo2ygenin, or dinitrophenyl, to which fluorescent antibodies can later be bound. %he probe and the chromosomes "from either the metaphase or interphase cells# that are being analy6ed are denatured and allowed to bind or hybridi6e to one another. If necessary, antibodies with a fluorescent tag are applied to the cells. %he cells are then viewed with a fluorescence microscope. %he fluorescent signals represent the probe"s# that is bound to the chromosomes

C !"#"$"#%& ' ("!) %. Each chromosome has many genes. &. (aired chromosomes segregate during meiosis. Each se) cell contains half the number of chromosomes as non"se) cells !somatic#. '. Chromosomes assort independently during meiosis. This means that the gamete receives one chromosome from each parent and that they do not influence each other.

Banding Patterns
Q-bands are like fluorescent G-bands, but certain heterochromatic regions are more brightly stained with Q-banding.