Chromosome Analysis

Karyotyping

process of preparing chromosomes for analysis

Carol Greider Elizabeth Blackburn Jack Szostak

Winners in triplicate

2009 Nobel Prize

On Christmas Day in 1984, Greider — then Blackburn's graduate student — saw the first evidence that this enzyme, which Greider and Blackburn named telomerase, was responsible for constructing telomere DNA

--Telomerase provides a platform enabling DNA polymerases to copy the entire length of the chromosome without missing the ends. Greider and Blackburn also showed that telomerase contains a key RNA sequence that acts as a template for the telomere DNA, which attracts proteins to form a protective cap around the ends of the DNA strands

Int. J. Cancer: 122, 1–4 (2008)

Int. J. Cancer: 122, 1–4 (2008)

Misidentity Ture identity

Int. J. Cancer: 122, 1–4 (2008)

Misidentity Ture identity

Int. J. Cancer: 122, 1–4 (2008)

Misidentity Ture identity

Oral Oncol. 2008 April ; 44(4): 369–382.

Chromosomal imbalances in oral squamous cell carcinoma.Examination of 31 cell lines and review of the literature

1. Cell line identification

2. Cell line characterization

3. Relating the species and sex they were derived

4. Distinguishing between normal and malignant cells

5. Identification of aberrant chromosome

6. Embryonic stem cell characterization

Karyotyping

Karyotype G-banding Technique for producing banding patterns in eukaryotic chromosomes Dark band , stained strongly with giemsa stain or acredine a. Heat hydrolysis

b. Trypsin treatment c. Giemsa at pH 9.0

R- banding Reverse banding ( Telomere bandinn) stain weakly with giemsa or acridine pretreatment with BaOH or NaOH followed by heat and salt

C-banding C (centromere or constitutive heterochromatin)

stains the heterochromatin in the centromeres

chromosomes 1, 9, and 16

Q- banding Quinacrine stain orange, fluorescent bands

G-banding

G banding

G-banding: Bands are produced by staining with Giemsa stain after pre treating chromosomes with trypsin

Each homologous chromosome pair has a unique pattern of G-bands, enabling recognition of particular chromosomes

Q- banding : fluorescent bands

Primary muscle cell culture

C-banding: stains the heterochromatin in the centromeres

Chromosome preparations

Materials:

culture of cells in log phase

colcemid, 10-5 M in PBSA

PBSA

Trypsin( 0.25%)

Hypotonic solution: 0.04M KCl, 0.025M sodium citrate

Acetic methanol fixative( glacial acetic acid : Methanol 1: 3 ) Giemsa stain ( stock diluted in buffer water pH 6.8-7.2)

Giemsa:

A polychromatic blood stain

Stain nucleus : pink

cytoplasm pale gray-blue

nucleoli: dark blue

Chromosome preparation

• Culture in log phase

• Add clocemid 1x 10-5M in 1: 100 vol ( final 1x10-7M) or 100ug final concentration

• After 4-6 hrs, remove medium gently, and add 0.25% trypsin, and incubate culture for 10 min

• Harvest cells by centrifugation, discard supernatant

• Resuspend cell in 5 ml hypotonic solution (KCl) and incubate 37oC, 15 min, varied for cell types ( do not exceed 15 min)

• Add equal volume of freshly prepared 1ml Acetic Methanol fixative solution, centrifuge 1200rpm, 10 min

• Discard supernatant, vortex cell , and add 1ml Acetic Methanol fixative solution slowly

• Drop the solution from 30cm height on to the cold slide, tilt the slid and spread the solution

• Dry over the slides over a beaker as it boiling( or in hot oven 62oC, exam the slides under microscope , and prepare more slides

• Stain the cells with Giemsa

Stainning with Giemsa

1. Immerse the slide in stain for 2 min

2. Place the dish into the water and allow the surplus stain to overflow from the top of the slide dish

3. Displace the remaining stain with running water