1. Chromosome Analysis Karyotyping
process of preparing chromosomes for analysis

2. Winners in triplicate Carol Greider Elizabeth Blackburn Jack Szostak
2009 Nobel Prize

4. 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

5. Int. J. Cancer: 122, 1–4 (2008)

6. Misidentity
Ture identity
Int. J. Cancer: 122, 1–4 (2008)

7. Misidentity
Ture identity
Int. J. Cancer: 122, 1–4 (2008)

8. Misidentity
Ture identity
Int. J. Cancer: 122, 1–4 (2008)

9. Chromosomal imbalances in oral squamous cell carcinoma.
Examination of 31 cell lines and review of the literature
Oral Oncol April ; 44(4): 369–382.

11. Karyotyping
Cell line identification
Cell line characterization
Relating the species and sex they were derived
Distinguishing between normal and malignant cells
Identification of aberrant chromosome
Embryonic stem cell characterization

12. G-banding Technique for producing banding patterns in eukaryotic
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

13. G-banding

14. G banding

15. 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

16. Q- banding : fluorescent bands

17. Primary muscle cell culture

18. C-banding: stains the heterochromatin in the centromeres

19. 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 )

20. Giemsa:
A polychromatic blood stain
Stain nucleus : pink
cytoplasm pale gray-blue
nucleoli: dark blue

21. 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

22. Stainning with Giemsa
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