Karyotyping and Chromosomal banding

Study Objective

• Study objective 1: To understand the structure of the chromosomes

• Study objective 2: to understand the various banding techniques and their application

Karyotype

• The karyotype is the complete chromosomal set of the nucleus of the cell. A diagrammatic representation of the karyotype with all of the pairs of chromosomes arranged in order of size iscalled an ideogram.

• Every organism has a standard karyotype, which provides a frame of reference for the analysis of mutations.

Karyotype (contd..)

• Karyotypic analysis is the study of all of the visible traits of chromosomes in a typical cell. This is useful in studying speciation.

• Let’s consider an example:Ex 1: distinct species of Drosophila that are very similar in appearance are distinguished easily by karyotypic analysis, which reveals numerous chromosomal inversions and translocations.

Chromosome structure

• Composed of chromatin, a combination of nuclear DNA and proteins

• For karyotyping cells are captured in metaphase

• metaphase stage in mitosis at which chromosomes are aligned along the cell equator

Chromosomal 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

chromosome breakage loss duplication or inverted segments.

Chromosomal Banding (contd..)• In the 1950s, 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,

• 16 and the acrocentrics (chromosomes whose centromeres are near the tips).

• The staining techniques used to make the bands visible were developed in the late 1960s and early 1970s.

Q-Banding Technique

• Developed by T. Caspersson and his colleagues• Quinacrine mustard, an alkylating agent, was the first

chemical to be used for chromosome banding• They observed alternating bright and dull bands

under fluorocent microscope• Quinacrine-bright bands were composed primarily of

DNA that was rich in the bases adenine and thymine • Quinacrine-dull bands were composed of DNA that

was rich in the bases guanine and cytosine

G-Banding Technique • Giemsa is the most commonly used stain in

cytogenetic analysis• Staining a metaphase chromosome with a

Giemsa stain is referred to as G-banding• Unlike Q-banding, most G-banding techniques

require pretreating• Chromosomes are pretreated with either salt

or a proteolytic (protein-digesting) enzyme.

G-Banding Technique (contd..)

• “GTG banding” refers to the process in which G-banding is preceded by treating chromosomes with trypsin.

• G-banding preferentially stains the regions of DNA that are rich in adenine and thymine.

• The regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light.

G-Banding Technique (contd..)

• 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.

G-banded metaphase from a normal female

G-banded karyotype from a normal female.

Yunis’s Technique

• Jorge Yunis introduced a technique to synchronize cells so they are held at the same stage in the cell cycle.

• Cells are synchronized by making them deficient in folate, thereby inhibiting DNA synthesis.

• By rescuing the cells with thymidine, DNA synthesis is initiated and the timing of the prophase and prometaphase stages of the cell cycle can be predicted.

• Yunis’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.

R-Banding Technique• R-banding is the reverse pattern of G bands • G-positive bands are light with R-banding method,

and vice versa• R-banding involves pretreating cells with a hot salt

solution that denatures DNA that is rich in adenine and thymine

• The chromosomes are then stained with Giemsa. • R-banding is helpful for analyzing the structure of

chromosome ends, since these areas usually stain light with G-banding.

C-Banding Technique

• C-banding stains areas of heterochromatin, which is tightly packed and repetitive DNA.

NOR-Staining

• NOR-staining, where NOR is an abbreviation for “nucleolar organizing region,”

• refers to a silver staining method that identifies genes for ribosomal RNA that were active in a previous cell cycle

Fluorescent in situ Hybridization (FISH)

• FISH is a molecular cytogenetic technique• Allows cytogeneticists to analyze chromosome

resolution at the DNA or gene level• FISH can be performed on dividing

(metaphase) and non-dividing (interphase) cells to identify numerical and structural abnormalities resulting from genetic disorders.

FISH (contd…)

• Utilizes Probes; there are 3 catogories1.Repetitive sequences, including alpha satellite

DNA, that bind to the centromere of a chromosome

2. DNA segments, representative of the entire chromosome, that will bind to and cover the entire length of a particular chromosome

3.DNA segments from specific genes or regions on a chromosome that have been previously mapped or identified.

FISH (contd…)• Probes are “Tagged” with fluorecent

nucleotides either directly or indirectly• This is done by attaching nucleotides to small

molecules such as biotin digoxygenin, or dinitrophenyl, to which fluorescent antibodies can later be bound

• The cells are then viewed with a fluorescence microscope. The fluorescent signals represent the probe(s) that is bound to the chromosomes

Chromosome Painting

References:

• Genetics by Richard Robinson, The Macmillan Science Library, Volume 1; Page 125-129.

• Encylopedia of Molecular Biology, Pg 2347.