Download Week 9th and 10th DNA isolation and Amplification

Week 9th and 10th
DNA isolation
and
Amplification
Isolating Recombinant Plasmids
Breaking the cell wall
Denaturing and Renaturing
Separation of plasmids
from other macromolecules
Escherichia coli
E.coli k-12
- Gram-negative bacteria
-straight rod shaped cells of
about 2 µm long and 0.5 µm wide,
-can grow and divide rapidly by binary
fission.
Morphology of E. coli cell
Cell wall structure (3 layers):
O157:H7
O111
1) lipopolysaccharide “outer membrane”
2) murein (peptidoglycan) middle layer
3) cytoplasmic membrane, inner layer
Quantifying Plasmid DNA
Quantify DNA using UV absorbance
260 nm
Nucleic acids
280 nm
Aromatic amino acids
ds DNA 50 ug/ml = absorbance of 1 at 260 nm
230 nm
peptide bond
DNA amplification by
Polymerase chain reaction
1983
PCR
Nobel prize
PCR
PCR
PCR versatility
•DNA amplification …
research fields
forensic fields
•Gene expression
•DNA sequencing
....
Avian brood parasitism
Finger printing
DNA Fingerprinting
Combined DNA Index System (CODIS)
1977
Walter Gilbert
DNA sequencing
Frederick Sanger
Nobel prize
1986
Leroy Hood
Automated sequencer
(Applied Biosystems Inc.)
Whole genome sequencing
1990-2000
Gel electrophoresis
Agarose
D-galactose
Agar is a linear polymer extracted from seaweed.
Agarose is obtained from purification of agar.
• Polymerized agarose is porous,
allowing for the movement of DNA
Scanning Electron
Micrograph of Agarose Gel
(1×1 µm)
3,6-anhydro
L-galactose
• DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the
positive pole (cathode).
• An agarose gel is used to slow the movement of DNA and separate by
size.
H2

O2

DNA
-
Power
+
DNA loading dye
Glyerol
xylene cyanol (blue green colored)
bromophenol blue (purple colored)
Orange G (orange colored)
Observation of Blood and Blood typing
The composition of the blood
Cells in Blood
Erythrocyte
(red blood cell)
Leukocyte
(white blood cell)
Platelets
Cells in Blood
Erythrocytes (Red blood cells)
Carry O2 and CO2
large surface to volume ratio: efficient gas exchange
Leukocytes (White blood cells)
Thrombocytes (Platelets)
Platelets
promote blood clotting, help repair gaps
in blood vessel walls
Blood Clotting
• When blood clots
– Fibrin traps red blood cells
– The yellowish liquid that separates from clotted blood is known as serum
•Serum contains a type of protein known as ANTIBODIES, which bind
specifically to one type of antigen
Antibodies
– They bind antigens to render them useless
– An antibody is tailor made to the antigen
Antigen-Antibody Binding
• Binding depends on 3-D structural match
between binding site and antibody
– Very Specific
• Ideally recognizes only 1 compound
Karl Landsteiner
1901 - Blood has different types
– Before this physicians tried to transfuse blood from one individual to another
often resulting in instantaneous death
– A – B – AB - O system of blood typing
– Nobel Prize in Physiology in 1930
4 Blood Types
• Type A
– Type A antigens on blood cells
– Anti-B antibodies in serum
• Type B
– Type B antigens on red blood cells
– Anti-A antibodies in serum
• Type AB (universal recipient)
– Both A and B antigens on red blood cells
– Neither Anti-A nor Anti-B antibodies
• Type O (universal donor)
– Neither A nor B antigens on red blood cells
– Both Anti-A and Anti-B antibodies in serum
Glycoproteins
type antigenpe
type antigenpe
Chromosome 9
O
A
B
"universal donor" is O Negative
"universal recipient" is AB Positive
erythroblastosis fetalis
ethidium bromide staining
Fragment size
10000
6000
4000
3000
2000
1000
bp
bp
bp
bp
bp
bp
2 μl
100
60
40
30
20
10
ng
ng
ng
ng
ng
ng
4 μl
200
120
80
60
40
20
ng
ng
ng
ng
ng
ng
8 μl
400
240
160
120
80
40
ng
ng
ng
ng
ng
ng