Karyotyping

Karyotyping

A test to examine chromosomes in a sample of cells. It can detect the genetic problem at the root of a disease. Karyotyping can be performed on almost any tissue, including amniotic fluid, blood, bone marrow and placental tissue.

C-banding

Identifies centromeres and heterochromatin. Treat chromosomes with acid, alkali and then g-band.

Before staining was developed

Before banding methods were developed, it was difficult to distinguish chromosomes through just the use of a light microscope. Cytologists developed banding methods by developing stains which would bind to DNA and generate characteristic banding patterns. This was a much more efficient and effective way to analyse the chromosomes.

Banding regions of g-banding

In general, heterochromatic regions, which tend to be AT-rich DNA and relatively gene poor, stain more darkly in G-banding. Less condensed chromatin regions tend to be GC-rich and transcriptionally active so they incorporate less giemsa and produce lighter coloured bands. G-banding produces reproducible patterns for each chromosome, and the patterns are shared between all individuals of a species.

Overview of process

Karyotyping is the process of pairing and ordering the chromosomes of an organism to provide a genome-wide snapshot of an individuals chromosomes. Karyotypes are prepared using standardised staining procedures that reveal characteristic structural features for each chromosome.

G-banding

Most commonly used banding method. G-banding uses giemsa dye and metaphase chromosomes are firstly treated with trypsin which is an enzyme which degrades proteins before the giemsa is applied. As trypsin partially digests some of the chromosomal proteins, the chromatin structure relaxes and the dye can access the DNA.

Q-banding

The first banding technique that was developed. Involves the use of fluorescent die quinacrine, which alkylates DNA and is subject to quenching over time. It was found that quinacrine produces characteristic and reproducible banding patterns for individual chromosomes. Q-banding is most commonly used to detect Y chromosome abnormalities or mosaicism.

Stage 1

The process begins by obtaining a short-term culture of cells from a specimen (i.e. blood). After a period of cell growth and multiplication, dividing cells are arrested in metaphase by the addition of colchicine, which is toxic to the mitotic spindles. The cells are then treated with a hypnotic solution which results in their nuclei swelling and bursting. The nuclei are then treated with a chemical fixative, dropped onto a glass slide and treated with various stains which reveal structural features of the chromosomes.

R-banding

X-chromosomal abnormalities, heat before staining with giemsa and light and dark bands are reversed.