Studies of cell
-Fractionation
-Purification/ Identification
-Structure/ Function
Organelle level
Cell fractionation
-Nucleus
-Mitochondria
-RER, cell membrane
-SER
-Cytosol
Cellular level
Microscope
Molecular level: Macromolecules
Proteins
Carbohydrates
Lipids
Nucleic acids
Atomic level
C, H, O, N, S, P
CONTENTS
Cell fractionation
Electrophoresis
Blotting and Hybridization
Polymerase Chain Reaction
DNA Sequences
Cell fractionation
A lab technique which uses a centrifuge to separate the
contents of a cell (organelles) into fractions, after the cell has
been gently lysed.
 The process to break the cells is “HOMOGENIZATION” and
the subsequent isolation of organelles is
“FRACTIONATION”.
 The centrifugation technique is employed to isolate
organelles regarding to their physical characteristics, e.g.,
size, shape and density. The methods frequently used are
“DIFFERENTIAL CENTRIFUGATION” and “DENSITY
GRADIENT CENTRIFUGRATION”.
HOMOGENIZATION
Cell lysis
HOMOGENIZATION
 Gently disrupt the cells to release cellular components
 Physical or non-physical cell lysis methods
Physical methods of cell disruption
 Disruption of cells results from the shearing forces generated between the cells and
either solid abrasive or liquid medium.
 Pastel and mortar homogenizer, Abrasive beads, Blender, Pressure
homogenization, Osmotic shock, Freezing/ thawing technique, Ultrasonification
Non-physical methods of cell disruption
 Organic solvents- to destroy membrane
 Chaotropic anions- to destabilize membrane
 Detergents- to dissolve proteins and lipids membrane
 Enzymatic digestion- to digest proteins, carbohydrates and lipids of cell wall
FRACTIONATION
Centrifugation
Cell Fractionation
Physical methods of cell disruption
1. Pastel and mortar homogenizer
2. Abrasive beads- sands, silica, alumina
3. Blender- special designed blades and chamber
4. Pressure homogenization- cells are imbibed with an inert gas (argon) which will form gas
bubbles inside cytoplasm when the cells are suddenly returned to atmospheric pressure, hence
rupture the membrane.
5. Osmotic shock- swelling and disrupting of cells in hypotonic solution
6. Freezing/ thawing technique- ice crystals rupture the cells
7. Ultrasonification- ultrasonic wave to break open the plasma membrane and leave the internal
organelles intact.
Cell Fractionation
Non-physical methods of cell disruption
1. Organic solvents- chloroform/methanol mixtures can dissolve membrane lipids
(destroy membranes) and release subcellular components.
2. Chaotropic anions- potassium thiocyanate, potassium bromide, lithium diiodosalicylate
act to destabilize lipid membranes consequently, the subcellular components are being
released.
3. Detergents- solubilize the integral membrane proteins by interacting with the
phospholipid bilayer, e.g., SDS (anionic), Deoxycholate (non-denaturing) and Triton X100 (non-ionic)
4. Enzymatic digestion- to digest proteins, carbohydrates and lipids of cell wall, Mixture
of enzymes: chitinases, pectinases, lipases, proteases, cellulases
Cell Fractionation
Homogenization medium
 Slightly hypo-osmotic or iso-osmotic – to preserve structural integrity of
organelles
 Osmoticums: sucrose, manitol, sorbitol
 Chelating agents: EDTA or EGTA (remove Ca2+ or Mg2+ which are
required by membrane proteases)
 Protease inhibitor: endopeptidases, exopeptidases
 The homogenization should be performed at 4oC to minimize protease
activity
Cell Fractionation
Fractionation
 The most widely used technique for fractionating cellular
components is centrifugation technique
 Particles of different density, size, and shape sediment at different
rate in a centrifugal field.
Factors affected the rate of sedimentation:
 particle size and shape
 the viscosity of suspending medium
 centrifugal field
* The particle remain stationary when the density of the particle and the density of
the centrifugation medium are equal
Cell Fractionation
Types of Centrifugation
1. Differential centrifugation
2. Rate-zonal centrifugation
3. Isopycnic centrifugation
A centrifuge working at speeds in
excess of 20,000 RPM is an
“ultracentrifuge”.
Cell Fractionation
Differential centrifugation
 Separates particles as a function of size and
density
 A particular centrifugal field is chosen over a
period of time
 Larger mass; lower centrifugation force;
lesser spin time
 Subjected to repeated steps with increasing
of centrifugation force
Differential centrifugation
Centrifugation force to pellet the cellular components
Cell components
Nucleus
Mitochondria, Lysosome
Chloroplast, Peroxisome
RER membrane
Cell membrane,
SER membrane
Ribosome
Cytosol fraction
Centrifugation force (x g)
800-1,000
20,000-30,000
50,000-80,000
80,000-100,000
150,000-300,000
Supernatant
Differential
Centrifugation
Pellet 1
Pellet 2
Pellet 3
Pellet 4
Rate-zonal Centrifugation
Rate-zonal centrifugation
centrifugation
 Medium
 Slightly viscous
 Density gradient; a positive increment in density
 e.g., sucrose, GuHCl
 Sample is applied on the top of density gradient
 Particles separate into a series of bands (zone) in
accordance to rate of sedimentation (S), size and shape
Molecules
separate
according
to size and
shape
Rate-zonal Centrifugation
Centrifugation
Fraction collection
Isopycnic Centrifugation
Isopycnic
centrifugation
 Based solely on the density of the particles
 Separation medium– self-generating density gradient medium (CsCl
medium)
 Unaffected by the size or the shape of the particles
 Mostly used to separate nucleic acids, large glycoproteins
What make rate-zonal and isopycnic centrifugations difference?
Electrophoresis
 Molecules are separated by electric force
 F = qE : where q is net charge, E is electric field strength
 The velocity is encountered by friction
 qE = fv : where f is frictional force, v is velocity
 Therefore, mobility per unit field (U) = v/q = q/f = q/6pr : where  is
viscosity of supporting medium, r is radius of sphere molecule
E
v
F
-
f
q+
+---
+
Electrophoresis
Factors affected the mobility of molecules
-
1. Molecular factors
• Charge
• Size
• Shape
2. Environment factors
• Electric field strength
• Supporting media (pore: sieving effect)
• Running buffer
+
Electrophoresis
Electrophoresis
Types of supporting media
 Paper
 Agarose gel (Agarose gel electrophoresis)
 Polyacrylamide gel (PAGE)
 pH gradient (Isoelectric focusing electrophoresis)
 Cellulose acetate
Agarose Gel
 purified large MW
polysaccharide (from agar)
 very open (large pore) gel
 used frequently for large DNA
molecules
Electrophoresis
Agarose gel staining
Ethidium bromide
Fluorescence dye
Pounseur-S dye
Electrophoresis
Polyacrylamide Gels
 Acrylamide polymer; very stable gel
 can be made at a wide variety of concentrations
 gradient of concentrations: large variety of pore sizes (powerful sieving effect)
Electrophoresis
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
 Sodium Dodecyl Sulfate = Sodium Lauryl
Sulfate: CH3(CH2)11SO3- Na+
 Amphipathic molecule
 Strong detergent to denature proteins
 Binding ratio: 1.4 gm SDS/gm protein
 Charge and shape normalization
Electrophoresis
Isoelectric Focusing
Electrophoresis (IFE)
- Separate molecules according to
their isoelectric point (pI)
- At isoelectric point (pI) molecule
has no charge (q=0), hence
molecule ceases
- pH gradient medium
Electrophoresis
2-dimensional Gel Electrophoresis
- First dimension is IFE (separated
by charge)
- Second dimension is SDS-PAGE
(separated by size)
- So called 2D-PAGE
- High throughput electrophoresis,
high resolution
2-dimensional Gel Electrophoresis
Spot coordination
 pH
 MW
Hybridization and
Blotting
Hybridization
Hybridization

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Can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily
degraded)
Dependent on the extent of complementation
Dependent on temperature, salt concentration, and solvents
Small changes in the above factors can be used to
discriminate between different sequences (e.g., small
mutations can be detected)
Probes can be labeled with radioactivity, fluorescent dyes,
enzymes, etc.
Probes can be isolated or synthesized sequences
Oligonucleotide probes



Single stranded DNA (usually 15-40 bp)
Degenerate oligonucleotide probes can be used to
identify genes encoding characterized proteins
• Use amino acid sequence to predict possible DNA
sequences
• Hybridize with a combination of probes
• TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) - could
be used for FWMDC amino acid sequence
Can specifically detect single nucleotide changes
Detection of Probes




Probes can be labeled with radioactivity, fluorescent dyes,
enzymes.
Radioactivity is often detected by X-ray film
(autoradiography)
Fluorescent dyes can be detected by fluorometers,
scanners
Enzymatic activities are often detected by the production of
dyes or light (x-ray film)
RNA Blotting (Northerns)



RNA is separated by size on a denaturing agarose gel and
then transferred onto a membrane (blot)
Probe is hybridized to complementary sequences on the
blot and excess probe is washed away
Location of probe is determined by detection method (e.g.,
film, fluorometer)
Applications of RNA Blots
Detect the expression level and transcript size of a
specific gene in a specific tissue or at a specific
time. Sometimes mutations do not affect coding
regions but transcriptional regulatory sequences
(e.g., UAS/URS, promoter, splice sites, copy
number, transcript stability, etc.)
Western Blot
Highly specific qualitative test
Can determine if above or below threshold
Typically used for research
Use denaturing SDS-PAGE
• Solubilizes, removes aggregates & adventitious proteins are
eliminated
Components of the gel are then transferred to a
solid support or transfer membrane
weight
Transfer
membrane
Paper towel
Paper towel
Wet filter paper
Western Blot

Block membrane e.g. dried nonfat milk
Rinse with ddH2O
Add monoclonal
antibodies
Rinse again
Antibodies will bind to specified protein
Add antibody against yours with a marker (becomes the antigen)
Stain the bound antibody for colour development
It should look like the gel you started
with if a positive reaction occurred
Polymerase Chain Reaction
(PCR)
PCR
 A simple rapid, sensitive and versatile in vitro method for selectively
amplifying defined sequences/regions of DNA/RNA from an initial complex
source of nucleic acid - generates sufficient for subsequent analysis and/or
manipulation
 Amplification of a small amount of DNA using specific DNA primers (a
common method of creating copies of specific fragments of DNA)
 DNA fragments are synthesized in vitro by repeated reactions of DNA
synthesis (It rapidly amplifies a single DNA molecule into many billions of
molecules)
 In one application of the technology, small samples of DNA, such as those
found in a strand of hair at a crime scene, can produce sufficient copies to
carry out forensic tests.
 Each cycle the amount of DNA doubles
Background on PCR
 Ability to generate identical high copy number DNAs made possible in
the 1970s by recombinant DNA technology (i.e., cloning).
 Cloning DNA is time consuming and expensive
 Probing libraries can be like hunting for a needle in a haystack.
 Requires only simple, inexpensive ingredients and a couple hours
 PCR, “discovered” in 1983 by Kary Mullis,
 Nobel Prize for Chemistry (1993).
 It can be performed by hand or in a machine called a thermal cycler.
Three Steps





Separation
Double Stranded DNA is denatured by heat into single strands.
Short Primers for DNA replication are added to the mixture.
Priming
DNA polymerase catalyzes the production of complementary new
strands.
Copying
The process is repeated for each new strand created
All three steps are carried out in the same vial but at different
temperatures
Step 1: Separation


Combine Target Sequence, DNA primers template, dNTPs,
Taq Polymerase
Target Sequence
1. Usually fewer than 3000 bp
2. Identified by a specific pair of DNA primers- usually oligonucleotides that are
about 20 nucleotides

Heat to 95°C to separate strands (for 0.5-2 minutes)
• Longer times increase denaturation but decrease enzyme and
template
Magnesium as a Cofactor
Stabilizes the reaction between:
• oligonucleotides and template DNA
• DNA Polymerase and template DNA
Heat
Denatures DNA by uncoiling the Double Helix strands.
Step 2: Priming

Primers anneal to the end of the strand
 0.5-2 minutes
 Shorter time increases specificity but decreases yield
Requires knowledge of the base sequences of the 3’ - end


Decrease temperature by 15-25 °
Selecting a Primer

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Primer length
Melting Temperature (Tm)
Specificity
Complementary Primer Sequences
G/C content and Polypyrimidine (T, C) or polypurine (A,
G) stretches
3’-end Sequence
Single-stranded DNA
Step 3: Polymerization



Since the Taq polymerase works best at
around 75 ° C (the temperature of the hot
springs where the bacterium was
discovered), the temperature of the vial is
raised to 72-75 °C
The DNA polymerase recognizes the
primer and makes a complementary copy
of the template which is now single
stranded.
Approximately 150 nucleotides/sec
Potential Problems with Taq
Lack of proof-reading of newly synthesized DNA.
 Potentially can include di-Nucleotriphosphates (dNTPs) that
are not complementary to the original strand.
 Errors in coding result
 Recently discovered thermostable DNA polymerases, Tth
and Pfu, are less efficient, yet highly accurate.

How PCR works
1. Begins with DNA containing a sequence to be amplified and a pair of
synthetic oligonucleotide primers that flank the sequence.
2. Next, denature the DNA at 94˚C.
3. Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s.
sequences flanking the target DNA.
4. Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq
polymerase derived from Thermus aquaticus).
5. Repeat the cycle of denaturing, annealing, and extension 20-45 times to
produce 1 million (220) to 35 trillion copies (245) of the target DNA.
6. Extend the primers at 70-75˚C once more to allow incomplete extension
products in the reaction mixture to extend completely.
7. Cool to 4˚C and store or use amplified PCR product for analysis.
Thermal cycler protocol Example
Step 1 7 min at 94˚C
Step 2 45 cycles of:
20 sec at 94˚C
20 sec at 64˚C
1 min at 72˚C
Step 3 7 min at 72˚C
Step 4 Infinite hold at 4˚C
Initial Denature
Denature
Anneal
Extension
Final Extension
Storage
The Polymerase Chain Reaction
PCR amplification


Each cycle the oligo-nucleotide primers bind most all
templates due to the high primer concentration
The generation of mg quantities of DNA can be
achieved in ~30 cycles (~ 4 hrs)
OPTIMISING PCR
THE REACTION COMPONENTS
Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root,
saliva, semen
 Thermo-stable DNA polymerase
e.g. Taq polymerase
 Oligonucleotides
Design them well!
 Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
RAW MATERIAL
 Organims, Organ, Tissue, cells ( blood, hair root, saliva, semen)
 Obtain the best starting material you can.
 Some can contain inhibitors of PCR, so they must be removed e.g. Haem
in blood
 Good quality genomic DNA if possible
 Blood – consider commercially available reagents Qiagen– expense?
 Empirically determine the amount to add
POLYMERASE
 Number of options available
Taq polymerase
Pfu polymerase
Tth polymerase
 How big is the product?
100bp
40-50kb
 What is end purpose of PCR?
1. Sequencing - mutation detection
-. Need high fidelity polymerase
-. integral 3’
2. Cloning
5' proofreading exonuclease activity
PRIMER DESIGN
Length ~ 18-30 nucleotides (21 nucleotides)
Base composition: 50 - 60% GC rich, pairs should have equivalent Tms
Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]
Initial use Tm–5°C
Avoid internal hairpin structures
no secondary structure
Avoid a T at the 3’ end
Avoid overlapping 3’ ends – will form primer dimers
Can modify 5’ ends to add restriction sites
PRIMER DESIGN
Use specific
programs
OLIGO
Medprobe
PRIMER
DESIGNER
Sci. Ed software
Also available on the internet
http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html
Mg2+ CONCENTRATION
1
1.5
2
2.5
3
3.5
4 mM
Normally, 1.5mM MgCl2 is optimal
Best supplied as separate tube
Always vortex thawed MgCl2
Mg2+ concentration will be affected by the amount of DNA, primers and
nucleotides
USE MASTERMIXES WHERE POSSIBLE
How Powerful is PCR?




PCR can amplify a usable amount of DNA (visible
by gel electrophoresis) in ~2 hours.
The template DNA need not be highly purified — a
boiled bacterial colony.
The PCR product can be digested with restriction
enzymes, sequenced or cloned.
PCR can amplify a single DNA molecule, e.g. from
a single sperm.
Applications of PCR


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
Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA,
etc.) for analysis
1. Gene isolation
2. Fingerprint development
Introduce sequence changes at the ends of fragments
Rapidly detect differences in DNA sequences (e.g., length) for identifying
diseases or individuals
Identify and isolate genes using degenerate oligonucleotide primers
• Design mixture of primers to bind DNA encoding conserved protein motifs
Genetic diagnosis - Mutation detection
basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10-9
bp
Applications of PCR
Paternity testing
Mutagenesis to investigate protein function
Quantify differences in gene expression
Reverse transcription (RT)-PCR
Identify changes in expression of unknown genes
Differential display (DD)-PCR
Forensic analysis at scene of crime
Industrial quality control
DNA sequencing
Sequencing of DNA by the
Sanger method