RESTRICTION DIGESTION

OF
HUMAN METAPHASE
CHROMOSOMES
By
Anita Swaminathan
Siddharth krishnan
Suchithra Seshadrinathan
G.Vimal
Guide : Dr.S.Meignanlakshmi
 Objectives
• Harvesting and culturing of Human Peripheral
Blood.
• Identify the most effective method to maximize
growth of lymphocytes
• Standardization of Karyotyping protocol to the
present lab conditions
• Obtain spreads of Human Metaphase
Chromosomes
• Restriction Digestion of Human Metaphase
Chromosomes
 Karyotyping
• Karyotyping is a diagnostic tool that allows us to
check the images of the structure and the
number of chromosomes in an individual’s
somatic cell. The preparation of an individual’s
Metaphase chromosomes sorted out by their
distinctive visual features is called a Karyotype.
• Any abnormalities in the chromosome number
or structure can be identified by comparing a
standard karyotype for that species.
• Obtaining results depends on various external
factors hence a protocol has to be standardized
for specific lab conditions
 Different methods tested for
obtaining the effective culture

• Whole Blood culture method

• Whole Blood Centrifugation
• Ficol-Paque Centrifugation Method
• RBC lysis Buffer Method
Harvesting and culturing of Human
Peripheral Blood.
• 5 ml of Whole blood was drawn and added
to a tube containing 40 microliters of
Heparin.
• 2.5 ml of Heparinized Blood was added to
autoclaved Centrifuge tube containing 7
ml of RPMI1640 medium and 3 ml of
filtered Bovine calf serum
• The tube is incubated in a normal
Incubator at 37 deg C for 72 hours.
Whole Blood Centrifugation Method
• 5 ml of Whole blood was drawn and added
to a tube containing 40 microliters of
Heparin.
• 2.5 ml of Heparinized Blood was
centrifuged at 2000 rpm for 15 minutes.
• Supernatant is innoculated into 7ml of
Medium with 3ml of Serum and incubated
at 37deg C for 72 hours.
Ficol-Paque Centrifugation Method
• 5 ml of Whole blood was drawn and added to a
tube containing 40 microliters of Heparin.
• 1.5ml of Balanced Salt Solution is added to 2.5
ml of Heparinized Blood.
• This mixture is added to 1.5ml of Ficol-Paque
and centrifuged at 2000 rpm for 15min
• White layer obtained which is rich in
lymphocytes is added to the fresh medium
• Incubation : 72hrs at 37 deg C
RBC lysis buffer method
 The follow-up procedure
• Culture was treated with 0.075M KCl for varying
time periods and centrifuged.
• Treated with Fixative (different fixatives were
tried) and centrifuged several times.
• cell solution is dropped using a pipette from a
minimum of 2ft height at angle of 45 deg on to a
slide for bursting of cells and cause spreading of
chromosomes.
• Slides are treated with Giemsa stain and viewed
under microscope.
 Results obtained ..
• Whole blood culture yielded the most
satisfactory spreads, but improvements are to
be made to reduce the amount of RBC’s so that
better spreads could be obtained.
• After several trials It was found that Kcl
treatment incubation for 1 hour was the optimum
incubation period to cause better spreads
• Geimsa staining for 1 hour produces better
stains.
• Different fixatives were tried and it was found
that methanol-acetic acid fixative was the most
effective.
 Work plan
• To try to obtain perfect chromosome
spreads
• To subject these spreads to restriction
digestion by Msp1 and Alu1