Chromosome banding

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Chromosomes display a banded pattern when treated with some stains. Bands are alternating light and dark stripes that appear along the lengths of chromosomes.

Unique banding patterns are used to identify chromosomes and to diagnose chromosomal aberrations, including chromosome breakage, loss, duplication, translocation or inverted segments.

A range of different chromosome treatments produce a range of banding patterns: Q-bands, G-bands, R-bands, C-bands, NOR-bands and T-bands.

In recent years a number of chromosome banding techniques have been developed that employ molecular cytogenetic techniques, for example fluorescence in situ hybridization (FISH).

Chromosome structure and bands

The chromosomes of eukaryotes are composed of a combination of nuclear DNA and proteins. During mitosis chromosomes replicate and condensed through coiling forming chromosomes consisting of two chromatids joined at the centromere. Each chromatid condenses approximately ten-thousand fold reaching maximal condensation at metaphase – DNA of roughly 5 cm in length is condensed to 5 micrometers in a metaphase chromosome. These condensed chromosomes are visible under the light microscope. 

During the condensation process DNA is looped around protein complexes called nucleosomes. This primary structure then undergoes more cycles of coiling producing the well-known metaphase chromosome structure. Some of the looped segments of DNA are close together and condense more than others, forming regions known as domains. These closely condensed domains tend to stain more darkly than the areas where the loops are more loosely arranged. 

In the order Diptera, which includes Drosophila, salivary gland chromosomes undergo repeated rounds of replication without cell division forming highly replicated chromosomes – polytene chromosomes. A polytene chromosome in a Drosophila salivary gland cell can contain as many as five thousand alternating dark and light bands. In these chromosomes the dark bands correspond to highly condensed domains and the lighter bands to less condensed DNA. It is in the less condensed areas where active genes can be identified. A gene becomes active by unravelling to permit transcription into messenger RNA. These unravelled regions are observed as “puffs” under the microscope. The gene becomes inactive by resolving the “puff” through condensation.  

Q-Banding

Quinacrine mustard, an alkylating agent, was the first chemical to band chromosomes viewed under a fluorescence microscope. Quinacrine dihydrochloride has subsequently been substituted by quinacrine mustard. The alternating bands of bright and dull fluorescence are called Q bands. The bright bands are primary composed of DNA rich in adenine and thymine, while the dull bands are rich in guanine and cytosine.

Q bands are especially useful for distinguishing the human Y chromosome and various chromosome polymorphisms involving satellites and centromeres of specific chromosomes.

G-banding

Giemsa has become the most commonly used stain in human cytogenetic analysis. Unlike Q-banding, G-banding usually requires pre-treating chromosomes with either salt or a proteolytic (protein-digesting) enzyme. When chromosomes are pre-treated with the proteolytic enzyme trypsin the process is called GTG banding. Giemsa stains preferentially regions rich in adenine and thymine. Therefore, G bands correspond closely to Q bands.

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.

R-banding

Reverse banding (R-banding) involves the incubation of slides containing metaphase chromosomes in hot phosphate buffer and stained with Giemsa. The banding pattern that results is essentially the reverse of G bands. R bands are GC-rich. The AT-rich regions are selectively denatured by heat leaving the GC-rich regions intact. Fluorochromes that are GC specific also produce a reverse chromosome banding pattern. R-banding is helpful for analyzing the structure of chromosome ends, since these areas usually stain light with G-banding.

C-Banding

C-banding stains areas of heterochromatin, which is tightly packed and repetitive DNA. C-banding is specifically useful in humans to stain the centromeric chromosome regions and other regions containing constitutive heterochromatin - secondary constrictions of human chromosomes 1, 9, 16, and the distal segment of the Y chromosome long arm.

NOR-banding

NOR-banding involves silver staining (silver nitrate solution) of the "nucleolar organizing region", which contains rRNA genes.

T-Banding

T-banding involves the staining of telomeric regions of chromosomes using either Giemsa or acridine orange after controlled thermal denaturation. T bands apparently represent a subset of the R bands because they are smaller that the corresponding R bands and are more strictly telomeric.

DAPI/Distamycin A Staining

DAPI/distamycin A fluorescent staining technique is a method for labelling a specific subset of C bands. DAPI/Distamycin A staining is useful in identifying peri-centromeric breakpoints in chromosomal rearrangements and in identifying chromosomes that are too small for standard banding techniques.