G and R Banding

G-banding is a technique used to produce a visible karyotype by staining condensed chromosomes. It is useful for identifying various genetic diseases through the photographic representation of the entire chromosome complement. The metaphase chromosomes are treated with trypsin (to partially digest the chromosome) and stained with Giemsa. Dark bands that take up the stain are strongly A,T rich (gene poor). The reverse of G-bands is obtained in R-banding.

It is difficult to identify and group chromosomes based on simple staining because the uniform color of the structures makes it difficult to differentiate between the different chromosomes. 

Giemsa's solution is a mixture of methylene blue, eosin, and azure B. It is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. Giemsa stain is used in Giemsa banding, commonly called G-banding, to stain chromosomes and often used to create an idiogram. It can identify chromosomal aberrations such as translocations and rearrangements.

limitations of this technique are the ineffectiveness of determining small translocations, detecting microdeletions, and characterizing the chromosomes of cell lines which are complex. However, it is a fast and low-cost technique to determine chromosome number, aneuploidy, large translocations, and macrodeletions.

A reverse Giemsa chromosome banding method that produces bands complementary to G-bands; induced by treatment with high temperature, low pH, or acridine orange staining.

Acridine orange was originally used to stain untreated chromosomes. Acridine orange (AO) is a base composition-independent fluorochrome that binds to DNA by intercalation and which gives relatively uniform fluorescence along the length of the chromosome arms. The dye binds very little to non-nucleic acid cell components, but it fluoresces orange-red when bound to single-stranded nucleic acids and yellow-green when bound to double-stranded nucleic acids. Following hot phosphate buffer treatment, R bands are yellow-green, and G/Q bands are orange-red. The major factor that contributes to R banding is the relative GC-richness of the R bands.