Skip to main content

Cytogenetics

G-banding

G-banding, technically known as GTG Banding (G-bands by Trypsin and Giemsa), is the most widely used routine staining technique in clinical cytogenetics in the United States. It provides a continuous pattern of light and dark bands along the entire length of the chromosome, allowing for the identification of each individual chromosome (1 through 22, X, and Y) and the detection of structural abnormalities such as translocations, deletions, and inversions. Unlike solid staining, which only allows counting and size grouping, G-banding creates a unique “fingerprint” for every chromosome region

Mechanism of Action

The G-banding pattern is the result of a differential interaction between the proteolytic enzyme (trypsin), the chromatin proteins (histones), and the DNA structure. The process relies on the fact that chromatin is not uniform; it is organized into domains with different functional and structural properties

  • Trypsin Digestion: Trypsin is a proteolytic enzyme that partially digests the proteins (histones and non-histone chromosomal proteins) that package the DNA. This digestion relaxes the chromatin structure and allows the dye to access the DNA. Crucially, the proteins in different regions of the chromosome have varying resistance to trypsin digestion
  • The Banding Pattern
    • Dark Bands (G-Positive): These regions stain intensely with Giemsa. They correspond to AT-rich DNA. Biologically, these areas are Heterochromatin - they are genetically inactive (gene-poor), replicate late in the S-phase of the cell cycle, and are more tightly condensed. The proteins in these regions are more resistant to trypsin digestion or the DNA structure retains the dye more effectively
    • Light Bands (G-Negative): These regions stain faintly or not at all (appearing white/pale). They correspond to GC-rich DNA. Biologically, these areas are Euchromatin - they are genetically active (gene-rich), replicate early in the S-phase, and are less condensed. The proteins here are more susceptible to digestion, or the open structure does not trap the dye
    • Note: The G-banding pattern is essentially the inverse of R-banding (Reverse banding)

The Step-by-Step Protocol

The G-banding procedure is a delicate balance. The goal is to digest the chromosomes just enough to reveal the bands, but not so much that the chromosome morphology is destroyed (making them look “fuzzy” or “ghost-like”)

  1. Slide Aging (Pre-treatment): Freshly prepared slides (straight from the harvest) do not band well. They are often too resistant to trypsin or produce irregular staining. Slides must be “aged” to stabilize the chromatin and evaporate residual water
    • Natural Aging: Leaving slides at room temperature for 3–7 days
    • Artificial Aging (Baking): Heating slides in an oven (e.g., \(60^\circ\text{C}\) for 18 hours or \(90^\circ\text{C}\) for 30–60 minutes) to simulate aging. This is the standard clinical workflow
  2. Trypsinization: The slide is immersed in a solution of Trypsin (often diluted in saline or buffer) for a specific time (seconds to minutes)
    • Variable: The activity of trypsin is temperature-dependent. Labs usually keep trypsin jars in a water bath or strictly controlled room temperature
  3. Rinsing: The enzymatic reaction is stopped immediately by dipping the slide in a rinsing solution (Isoton, Saline, or Buffer). This prevents over-digestion
  4. Staining: The slide is immersed in Giemsa Stain (or Leishman/Wright stain). The stain is typically diluted in a phosphate buffer to maintain a specific pH (6.8)
  5. Rinsing & Drying: Excess stain is washed off with distilled water, and the slide is air-dried before coverslipping

Critical Variables & Optimization

G-banding is often described as an “art” because the optimal conditions fluctuate based on the environment and the sample

  • Slide Aging vs. Trypsin Time: There is an inverse relationship between slide age and trypsin sensitivity
    • Fresh Slides: The chromatin is soft. They require very short: trypsin exposure. If trypsinized too long, they will disintegrate (“ghosts”)
    • Old Slides: The chromatin is hard/oxidized. They require longer: trypsin exposure to reveal bands. If under-trypsinized, they will look solid-stained (no bands)
  • pH Control (The Buffer): The staining reaction is highly pH-sensitive
    • Optimal pH (6.8): Produces the classic purple/magenta chromosome with sharp contrast
    • Too Acidic (<6.4): Chromosomes appear red/pink. Bands are faint or non-existent
    • Too Basic (>7.2): Chromosomes appear dark blue/black. The stain is too heavy, obscuring the bands
  • Trypsin Strength
    • Under-Trypsinized: The chromosomes appear solidly dark (like a solid stain). No distinct bands are visible. The enzyme did not relax the chromatin enough
    • Over-Trypsinized: The chromosomes look “puffy,” “fuzzy,” or “melted.” The telomeres may look ragged. In severe cases, “empty” chromosomes (ghosts) are seen where only the outline remains

Resolution Levels (ISCN Standards)

The quality of G-banding is measured by the number of bands visible in a haploid set of chromosomes. This resolution is determined primarily by the harvest (chromosome length), but the banding technique must match the resolution

  • 400-Band Level: Routine resolution. Sufficient for counting chromosomes (aneuploidy) and identifying large translocations. Typically seen in standard metaphase cells
  • 550-Band Level: Standard clinical resolution. Allows for the detection of smaller deletions and rearrangements. Requires late-prophase/early-metaphase chromosomes
  • 850-Band Level: High-Resolution. Required for microdeletion syndromes. Requires prometaphase chromosomes (very long)
    • Banding Challenge: High-resolution chromosomes are very sensitive to trypsin. The laboratory scientist must reduce the trypsin time significantly to avoid destroying the fine sub-bands