CH 18

CHAPTER 18
Methods in Cell Biology
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Keys
Describe types of information that can be obtained by using various techniques.

Define magnification and resolution for light microscopy.
Describe components of the light and electron microscopes and their functions.
Outline different light and electron microscopy techniques emphasizing the
information they reveal and their advantages and disadvantages.
Outline the factors that cause the formation of different radioisotopes and
describe the major methods used to detect radioactivity.
Describe cell culture techniques and advantages of working in that system.
Discuss various methods used to purify organelles, proteins, and nucleic acids
and the traits of these molecules by which the methods effect purification.
Outline nucleic acid hybridization techniques and recombinant DNA technology.
Discuss polymerase chain reaction and techniques for DNA sequencing.
Discuss the production and use of antibodies in research.
Introduction
Research in cell biology requires complex instrumentation and
techniques.
Cell and molecular biology is more dependent on the development of
new instruments and technologies than any other branch of biology.
Understanding the technology helps in understanding the cell.
It is important to understand these techniques and the types of
information that can be learned by using these techniques.
(18.1) The Light Microscope

Paths taken by light
rays that form the
image of the
specimen and those
that form the
background light of
the field.
Sectional diagram
through a compound
light microscope that
has both an objective and
an ocular lens
The light microscope uses the refraction of light rays to

magnify an object.
A condenser directs light toward the specimen.
The objective lens collects light from the specimen.
The ocular lens forms an enlarged, virtual image.
The Light Microscope

Resolution
Magnification versus
resolution: (a) to (b) has
increased magnification and
resolution, but (b) to (c) only
increased magnification
Resolution
Relationship between the

stimulation of individual
photoreceptors (left) and the
resulting scene one would perceive
(right).
Resolution is the ability to see two nearby points as distinct images.

The numerical aperture is a measure of the light-gathering qualities of a
lens.
The limit of resolution depends on the wavelength of light.
Optical flaws, or aberrations, affect resolving power.

Visibility
Visibility
Visibility deals with factors
that allow an object to be
observed.
It requires that the specimen
and the background have
different refractive indexes.
Translucent specimens are
stained with dyes.
A bright-field microscope
a light that illuminates the
specimen is seen as a bright
background; it is suited for
specimens of high contrast
such as stained sections of
tissues.
Feulgen stain: specific for

DNA, showing the
chromosomes of an onion
root tip cell in metaphase of
mitosis at the time it was
fixed.

Visibility
Preparation of
Specimens for BrightField Light
Microscopy
A whole mount is an
intact object, either
living of dead.
A section is a very
thin slice of an object.
Feulgen stain: specific for

To prepare a section,
DNA, showing the
cells are immersed in
chromosomes of an onion
a chemical called a
root tip cell in metaphase of
fixative.
mitosis at the time it was
The rest of the
fixed.
procedures minimize
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John Wiley
alteration
from
the & Sons, Inc. All rights reserved.

Phase-contrast
Phase-Contrast
Microscopy
The phase-contrast
microscope makes
highly transparent
objects more visible by
converting differences in
the refractive index of
some parts of the
specimen into differences
in light intensity.
Differential interference
contrast (DIC) optics
gives a three-dimensional
quality to the image.
A comparison
of cells seen
with different
types of light
microscopes:
a ciliated
protist under
bright-field
(top), phasecontrast
(middle), and
differential
interference
contrast
(bottom).

Fluorescence microscopy
Fluorescence Microscopy (and Related Fluorescence-Based

Techniques)
Fluorescence microscopy has made possible advances in livecell imaging.
Fluorochromes are compounds that release visible light upon
absorption of UV rays.
Fluorochrome stains cause cell components to glow, a
phenomenon called fluorescence.
Fluorochrome-conjugated antibodies are used to locate specific
cellular structures (immunofluorescence).
The gene for green fluorescent protein (GFP) from jellyfish can
be recombined with genes of interest in model organisms.
GFP is expressed with the host gene of interest.
GFP is used to follow a gene of interest.

Use of GFP variants to
follow the dynamic
interactions between
neurons and target cells in
vivo: portion of a transgenic
mouse brain with two
differently colored
fluorescent neurons.
Fluorescence Microscopy
The gene for green fluorescent protein (GFP) from jellyfish can
be recombined with genes of interest in model organisms.
GFP is expressed with the host gene of interest.
GFP is used to follow a gene of interest.

FRET
A GFP variant is called
fluorescence resonance energy
transfer (FRET), which uses
fluorochromes to measure
changes in distance between
labeled cellular components.
Proteins or individual domains
can be fused with different GFP
variants.
Energy is transferred from one
GFP variant to the other GFP
variant.
Fluorescence resonance
energy transfer (FRET). This
diagram shows an example of the
use of FRET technology to follow
the change in conformation of a
protein (PKG) following cGMP
binding.

Video microscopy
Video Microscopy and Image Processing
Video microscopy is used to observe living cells.
Video cameras offer several advantages for viewing specimens.
Special types of video cameras (called charge-coupled device, or CCD
cameras) are constructed to be very sensitive to light, which allows them to
image specimens at very low illumination.
They can detect and amplify very small differences in contrast.
Images produced by video cameras can be converted to digital electronic
images and processed by a computer.
In one technique, the distracting out-of-focus background in a visual field is
stored by the computer and then subtracted from the image containing the
specimen, greatly increasing the clarity of the image.

Laser scanning confocal microscopy
The light
paths in a
confocal
fluorescenc
e
microscope
and the
focal plane
Confocal fluorescence micrographs

of three separate optical sections,
each 0.3 m thick, of a yeast
nucleus stained with two different
fluorescently labeled antibodies
(DNA in red and a telomere-binding
protein in green).
Laser Scanning Confocal Microscopy
A laser scanning confocal microscope produces an image of a
thin plane located within a much thicker specimen.
A laser beam is used to examine planes at different depths in a
specimen.

STORM
Breaking the light microscopes limit of

resolution: conventional fluorescence micrograph
(left) and STORM micrographs. Shown are microtubules
(green) and clathrin-coated pits (red).
Super-Resolution Fluorescence Microscopy
STORM (stochastic optical reconstruction microscopy) allows the

localization of a single fluorescent molecule within a resolution of <20 nm.
Fluorescent images can be positioned with greater accuracy.
(18.2) Transmission Electron

Microscope
Transmission electron
microscopes (TEMs)
use electrons instead
of light to form images.
The limit of resolution
is about 10-15 .
The resulting images
display a 100- to 200fold increase in
resolution over
traditional light
microscopy.
Increased amount of
information at the
subcellular level.
Comparison of skeletal muscle

images from a light (top) and
electron (bottom) microscope at a
comparable magnification of 4500
times actual size.
Transmission Electron Microscope
The components of an
electron microscope:
An electron beam from a
tungsten filament
accelerated by high voltage,
and focused with a magnetic
field.
A condenser lens is placed
between the electron source
and the specimen.
Differential scattering of
electrons by the specimen
creates the image.
Proportional to the
thickness of the
specimen.
Tissues are stained with
heavy metals for contrast.
A comparison of the lens

systems of a light and
electron microscope.
Transmission Electron Microscopy

Specimen preparation
Specimen Preparation for
Electron Microscopy
Specimens must be fixed,
embedded, and sectioned
thinly.
Glutaraldehyde and
osmium tetroxide are
common fixatives.
Specimens are
dehydrated prior to
embedding.
Epon or Araldite are
common embedding
resins.
Thin sections cut with
glass or diamond knives
are collected on grids.
Preparation of a specimen for

observation in the electron
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reserved.

Negative staining & shadow casting
EM: tobacco
rattle virus after
negative
staining with
potassium
phosphotungstat
e (top) or
shadow casting
with chromium
(bottom).
The procedure used for

shadow casting as a
means to provide
contrast in the electron
microscope
Chemicals used may cause artifacts, which may be disproved with other techniques.
In negative staining, heavy metal diffuses into spaces between specimen molecules.
Shadow casting coats a specimen with metal to produce a three-dimensional effect.

Freeze-fracture replication and etching
Freeze-Fracture
Replication and FreezeEtching
In freeze-fracture
replication, frozen
tissue is fractured with
a knife.
A heavy-metal layer
is deposited on
fractured surface.
A cast of the surface
is formed with
carbon.
The metal-carbon
replica is viewed in
the TEM.
In freeze-etching, a
layer of ice is
evaporated from
the
Procedure for
the formation
of freezefracture
replicas and
freeze-etching

Freeze-fracture replication and etching
Freeze-Fracture
Replication and FreezeEtching
In freeze-fracture
replication, frozen tissue
is fractured with a knife.
A heavy-metal layer is
deposited on fractured
surface.
A cast of the surface is
formed with carbon.
The metal-carbon replica
is viewed in the TEM.
In freeze-etching, a
layer of ice is evaporated
from the surface of the
specimen prior to coating
it with heavy metal.
Replica of a
freeze-fractured
onion root cell:
nuclear envelope
(N.E.) and pores
(N.P.), Golgi (G),
vacuole (V), and cell
wall (C.W.).
Deep
etching: EM
of ciliary
axonemes
from the
protozoan
Tetrahymena
.
(18.3) Scanning Electron Atomic

Force Microscopy
Scanning electron
microscopes (SEMs)
form images from
electrons bounced off
the specimen surface.
Specimens for SEM are
dehydrated by criticalpoint drying then
coated with a layer of
carbon, then gold.
The image in SEM is
indirect.
SEM has a wide range
of magnification and
focus.
SEMs: (left) T4 bacteriophage (X

275,000) and (right) the head of
an insect (X 40).
Scanning Electron Atomic Force

Microscopy
Atomic Force Microscopy (AFM)

AFM is a high-resolution
scanning instrument.
AFM provides an image of
each individual molecule as it
is oriented in the field.
A limitation of electron
microscopy and X-ray
crystallography is that they
proved snapshots.
AFM can obtain rapid
sequential images of a
macromolecule so that its
activity can be followed over
real time.
High-speed atomic
force microscopy:
Forces between the
sample and the AFM tip
cause deflections of
the tip, which are
detected by a laser
beam.
(18.4) The Use of Radioisotopes
Radioisotopes can be easily

detected and quantified.
Properties of radioisotopes:
An isotope refers to atoms
that differ in the number
of neutrons.
Isotopes with an unstable
combination of protons
and neutrons are
radioactive.
The half-life of a
radioisotope measures its
instability; half of the
radioactive material
disintegrates in a given
amount of time.
Properties of a variety of
radioisotopes
The Use of Radioisotopes
Radioisotopes can be
easily detected and
quantified.
Liquid scintillation
spectrometry
Scintillants absorb the
energy of an emitted
particle and release it in
the form of light.
Radiation of a tracer in a
sample can be detected
by measuring light
emitted by a scintillant.
Properties of a variety of
radioisotopes
The Use of Radioisotopes

Autoradiography
Autoradiography is
a technique to where
a particular isotope is
located.
A particle emitted
from a radioactive
atom activates a
photographic
emulsion.
The location of the
radioisotope in the
specimen is
determined by the
positions of the
overlying silver grains
in a photographic
emulsion.
Preparation of
a light
microscopic
autoradiograp
h.
(18.5) Cell Culture

Most of the study of cells is carried out using cell
culture.
Cells can be obtained in large quantities.

Most culture contain a single type of cell.
Many different types of cells can be grown in culture.
Cell differentiation can be studied in a cell culture.
Cells in a culture require media that includes hormones

and growth factors.
A primary culture is when cells are obtained directly
from the organism.
A secondary culture is derived from a previous culture.
A cell line refers to cells with genetic modifications that
allow them to grow indefinitely.
Many types of plant cells can be grown in culture.
Cell Culture
2D vs 3D
Comparison of the
morphology of cells
growing in 2D versus
3D cultures: (left)
fibroblast in 2D culture is
highly flattened with
broad lamellipodia, (right)
fibroblast in 3D matrix is
non-flattened, with a
spindle-shaped
morphology. White:
A two-dimensional culture system is when
cells are
grown
on
adhesion
sites;
blue:
fibronectin matrix.
the flat surface of a dish.
Labs are moving to three-dimensional cultures in which cells

are grown in a 3D matrix consisting of extracellular materials.
3D cultures are better suited to study cell-cell interactions.
(18.6) The Fractionation of a Cells

Contents by Differential
Centrifugation
Differential
centrifugation facilitates
the isolation of particular
organelles in bulk quantity.
Prior to centrifugation,
cells are broken by
mechanic disruption in a
buffer solution.
The homogenate is
subjected to a series of
sequential
centrifugations.
Organelles isolated can be
used in a cell-free system
to study cellular activities
Purification of subcellular
fractions by density-gradient
equilibrium centrifugation: in a
continuous sucrose-density gradient
different organelles sediment
according to density, where they
form bands.
(18.7) Isolation, Purification, and

Fractionation of Proteins
Protein purification involves the stepwise removal
of contaminants.
Purification is measured as an increase in specific
activity of a protein.
Some identifiable feature of the specific protein must be
utilized as an assay to determine the relative amount of
the protein.
Selective Precipitation
At low ionic strength, proteins tend to remain in solution.
At high ionic strength, protein solubility decreases.
Ammonium sulfate is the most commonly used salt for
protein precipitation.
Isolation, Purification, and Fractionation

of Proteins
Liquid column chromatography
Liquid Column Chromatography
Chromatography includes a variety of techniques in
which a mixture of dissolved components is
fractionated through a porous matrix.
Components are fractionated between mobile and
immobile phases.
The greater the molecules affinity for the matrix,
the slower its movement.
High performance liquid chromatography (HPLC)
has greater resolution due to a tightly packed
matrix.

of Proteins
Ion-exchange chromatography
Ion-exchange
chromatography uses ionic
charge as a basis for
purification.
The overall charge of a protein is a
summation of all the individual
charges of its component amino
acids.
A pH when the number of positive
and negative charges is equal is
the isoelectric point.
DEAE-cellulose is positively
charged and therefore binds
negatively charged molecules; it is
an anion exchanger.
CM-cellulose is negatively charged
and acts as a cation exchanger.
Ion-exchange
chromatography: separation of
two proteins by DEAE-cellulose
which binds the negatively
charged protein.

of Proteins
Gel filtration chromatography
Gel filtration separate

proteins by molecular
weight.
A column is packed with
cross-linked
polysaccharides of different
porosity.
Proteins small enough to
enter the pores are eluted
last.
Larger proteins unable to
enter the beads remain
dissolved in the moving
solvent phase.
Among those proteins that
enter the beads, smaller
species are slowed to a
greater extent than larger
ones.
Gel filtration chromatography:

separation of three globular
proteins having different
molecular mass. Among proteins
of similar basic shape, larger
molecules are eluted before
smaller molecules.

of Proteins
Affinity chromatography
Affinity chromatography
isolates one protein from a
mixture using a specific
ligand.
The technique can achieve
near-total purification in a
single step.
Proteins interact with
specific substances: e.g.
enzymes with substrates,
receptors with ligands,
antigens with antibodies.
Use column in which the
specific interacting
molecule is covalently
linked to an inert,
immobilized material.
Affinity chromatography:
Schematic of the coated agarose
beads to which only a specific
protein can combine (top) and steps
in the chromatographic procedure
(bottom).

of Proteins
Yeast two-hybrid
Determining Protein-Protein
Interactions
Antibodies establish protein
interactions by coprecipitation.
The yeast two-hybrid
system:
A DNA binding domain is linked
to the gene for one protein
the bait protein.
An activation domain is linked
to genes encoding possible
proteins that interact with the
bait.
A reporter gene (lac Z) is only
expressed when the bait and
its partner interact.
Use of the yeast two-hybrid system.


of Proteins
Polyacrylamide Gel Electrophoresis
Polyacrylamide Gel
Electrophoresis
Electrophoresis is based on
the migration of proteins in an
electric field.
In polyacrylamide gel
electrophoresis (PAGE),
proteins are driven through a gel
matrix.
Movement of proteins depends
on molecular size, shape, and
charge density.
The progress of the gel can be
followed using a charged
tracking dye.
The positions of the proteins can
be visualized through
autoradiography or Western blot.
Polyacrylamide gel electrophoresis


of Proteins
Polyacrylamide Gel Electrophoresis
Polyacrylamide Gel
Electrophoresis
Electrophoresis is based on
the migration of proteins in an
electric field.
SDS-PAGE
It is PAGE carried out in the
presence of a charged
detergent, sodium dodecyl
sulfate (SDS).
The repulsion between
bound SDS molecules
causes the proteins to unfold
into a similar shape.
Proteins become separated
solely on the basis of mass.
Polyacrylamide gel electrophoresis


of Proteins
2D Gel Electrophoresis
Two-Dimensional Gel
Electrophoresis
It separates proteins on the
basis of both isoelectric
focusing and molecular
weight.
After separation by isoelectric
focusing, the gel is removed
and subjected to SDS-PAGE.
Proteins can then be analyzed
mass spectrometry.
The technique is ideal for
detecting changes in the
proteins in a cell under
different conditions.
Two-dimensional gel
electrophoresis: HeLa cell
non-histone chromosomal
proteins labeled with
[35S]methionine resolving over
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reserved.
1,000
different proteins.

of Proteins
Spectrometry
Protein Measurement and Analysis

The amount of protein can be
determined measuring the amount
if light absorbed using a
spectrophotometer.
Mass spectrometry (MS) measures
the mass of molecules, determines
chemical formulas and molecular
structure, and identifies unknown
substances.
Protein fragments are converted to
ions and separated on the basis of
mass and charge.
Fragments are compared to large
protein databases for identification.
Principles of
operation of a
mass
spectrometer
(18.8) Determining the Structure

of Proteins and Multisubunit
Complexes
X-ray diffraction
analysis.
Schematic
diagram of the
diffraction of Xrays by atoms of
one plane of a
crystal onto a
photographic
plate
X-ray crystallography (or X-ray diffraction) uses protein crystals.
Crystals are hit with X-rays, and scattered radiation is collected on a

photographic plate.
The diffraction pattern provides information about the structure of a protein.
The technique is useful in the study of both proteins and nucleic acids.
Determining the Structure of

Proteins and Multisubunit
Complexes
Information gathered for a protein depends on the
resolution
In myoglobin, 6 resolution shows how polypeptide chain is
folded and the location of the heme moiety.
At 2 resolution, groups of atoms can be separated from
one another.
At 1.4 , the positions of individual atoms can be
determined.
To date, the structures of several hundred proteins have
been determined at atomic resolution (1.2 ) and a few as
low as 0.66 .
Electron density distribution of a

small organic molecule
(diketopiperazine) calculated at
several levels of resolution.
Determining the Structure of

Proteins and Multisubunit
Complexes
Alternate techniques:
In electron cryomicroscopy particles are placed on a
grid and rapidly frozen in a hydrated state in liquid
nitrogen without being fixed or stained.
X-ray crystallography and electron microscopic
reconstructions to show how the individual molecules
that make up a multisubunit complex interact.
Electron crystallography analysis of frozen specimens
can also be utilized in the study of membrane
proteins in the lipid bilayer.
Combining data from electron

microscopy and X-ray
crystallography: reconstruction
of an actin-ADF filament. Actin
(red) and ADF molecules (green).
(18.9) Fractionation of Nucleic

Acids
DNA fragments in a gel can be

revealed by immersing the gel in an
ethidium bromide solution and then
viewing by ultraviolet light
Separation of DNA by gel electrophoresis.
PAGE is used for separation of small DNA
and RNA molecules; large ones are
separated by agarose.
Nucleic acids are separated on the basis of
molecular weight.
Gel electrophoresis to
separate DNA
restriction fragments
by size
Fractionation of Nucleic Acids

Sedimentation
Ultracentrifugation
Velocity Sedimentation: rate at
which a molecule moves in
response to centrifugal force.
Size of organelles and
macromolecules expressed in
S (Svedberg) units.
The S value provides a good
measure of relative size.
Equilibrium Centrifugation
separates nucleic acids based
on their buoyant density.
Sensitive enough to separate
DNA molecules by base
composition.
Separation
of differentsized DNA
molecules by
velocity
sedimentatio
n
Separation of DNA molecules by

equilibrium sedimentation on the
basis of differences in base
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Fractionation of Nucleic Acids

Sedimentation
Ultracentrifugation
Velocity Sedimentation:
rate at which a
molecule moves in
response to centrifugal
force.
Size of organelles and
macromolecules
expressed in S
(Svedberg) units.
The S value provides
a good measure of
relative size.
Fractionating a tubes contents
Equilibrium
into successive aliquots allows
Centrifugation
for the absorbance of the
separates nucleic acids
solution in each fraction to be
based on their buoyant
measured and plotted.
density.
(18.10) Nucleic Acid Hybridization
Determining the
location of specific
DNA fragments in
a gel by a
Southern blot
Nucleic acid hybridization is based on the ability of two

complementary DNA strands to form a double-stranded hybrid.
The Southern blot technique is based upon DNA hybridization.
The Northern blot technique is based upon RNA-DNA hybridization.
Hybridization can be used to determine the degree of similarity
between two samples.
(18.11) Chemical Synthesis of DNA

Chemical synthesis of DNA or RNA supports many other procedures.
Hybridization analysis requires single-stranded nucleic acid
molecules for use as probes.
The chemical reaction linking nucleotides have been automated.
A nucleotide is assembled one at a time up to a total of 100
nucleotides.
Modifications (biotin and fluorophores) can be incorporated into the
molecules.
If a double-stranded molecule is needed, it is synthesized as two
complementary single strands that can be hybridized together.
Longer synthetic molecules are made in segments which are joined
together.
(18.12) Recombinant DNA

Technology
Recombinant DNA
molecules contain DNA
sequences derived from more
than one source.
Restriction endonucleases
are enzymes that function in
bacteria to destroy viral DNA,
restricting the growth of
viruses.
Are used to dissect genomes
into precisely defined fragments
for further analysis.
Restriction maps are complete
diagrams of the fragments that
result from digestion of a
genome by specific restriction
enzymes.
Restriction map construction: small

circular genome of the DNA tumor
virus polyoma
Recombinant DNA Technology

Formation of Recombinant DNAs
DNA is first cut with restriction
enzymes.
Recombinant DNAs can be
formed in various ways, such as
creating sticky ends with
restriction enzymes.
Two components of a
recombinant DNA are linked by
DNA ligase.
DNA cloning is a technique to
produce large quantities of a specific
DNA segment.
DNA segment to be cloned is first
linked to a vector DNA.
Bacterial plasmids and viruses
are two commonly used vectors.
Formation of a
recombinant
DNA molecule

Cloning
Cloning Eukaryotic DNAs
in Bacterial Plasmids
Plasmids used for DNA
cloning are modified forms of
the wild type.
Cloning plasmids contain a
replication origin.
Cloning plasmids usually
carry genes for antibiotic
resistance.
Example of
DNA
cloning with
bacterial
plasmids
Recombinant plasmids are

introduced into bacterial
cells by transformation.
Plasmid-containing bacteria
are selected by treatment
with antibiotics.

Cloning
Cloning using plasmids
Cells containing various
plasmids are grown into
separate colonies which can
be screened for the
presence of a particular
DNA sequence.
Replica plating produces
dishes containing
representatives of the same
bacterial colonies in the
same position in each dish.
In situ hybridization uses a
labeled DNA probe to locate
the colony having the
desired DNA fragment.
Replica plating
procedure to produce
identical copies of the
original bacterial plate

Cloning
Cells containing various plasmids are grown into separate
colonies which can be screened for the presence of a
particular DNA sequence.
Replica plating produces dishes containing representatives
of the same bacterial colonies in the same position in each
dish.
In situ hybridization uses a labeled DNA probe to locate the
colony having the desired DNA fragment.
Locating a bacterial colony
containing a desired DNA
sequence by in situ
hybridization

Cloning
Once the colony has
been identified, live
cells from the colony
can be grown into large
colonies to amplify the
recombinant DNA
plasmid.
The cells can then be
harvested, the DNA
extracted and the
recombinant plasmid
DNA separated from
the larger chromosome
by equilibrium
centrifugation.
Separation of plasmid DNA

from that of the main bacterial
chromosome by CsCl
equilibrium centrifugation

Cloning
Cloning Eukaryotic DNAs
in Phage Genomes
Recombinant DNA
molecules are packaged
into lambda phage heads.
Lambda phage can take
inserts up to a size of 25
kb.
Sites of phage infection
are identified as plaques in
a bacterial lawn.
DNA fragments are
identified by the same
techniques as those used
for cloning of recombinant
plasmids.
Protocol for cloning

eukaryotic DNA fragments
in lambda phage
(18.13) Enzymatic Amplification of DNA

by PCR
Polymerase chain
reaction (PCR) is a
technique to amplify specific
DNA fragments.
It uses a very small amount of
template.
Utilizes a heat-stable DNA
polymerase (Taq polymerase)
from bacteria living in hot
springs.
Uses repeated cycles of
denaturation, DNA replication,
and cooling to double the
amount of DNA during each
cycle.
Uses an automated thermal
cycler.
Polymerase chain reaction (PCR)

Enzymatic Amplification of DNA by PCR

Applications
Applications of PCR
Amplifying DNA for cloning or analysis
For criminal investigations, select regions of the genome for
amplification that are highly polymorphic so that no two people
will have the same results.
Fossil analysis from samples millions of years old.
Testing for the presence of specific DNA sequences

Determine whether or not a tissue sample contains a particular
virus.
Comparing DNA molecules

Quick assays that compare the similarity of two DNA samples
such as genomic DNA from bacterial isolates.
Quantifying DNA or RNA templates

The rate of accumulation of product is proportional to the amount
of template present in the sample.
(18.14) DNA Sequencing

Techniques developed in the 1970s are
widely used for sequencing nucleic
acids.
The Sanger-Coulson dideoxy method
became the most widely used:
Four samples of identical singlestranded DNA molecules are
obtained.
DNA of each sample is incubated with
a primer, DNA polymerase, four
dNTPs, and a low concentration of
ddNTPs (dideoxyribonucleoside
triphosphates), different one in each
sample.
DNA fragments of different lengths
are synthesized in each sample, with
synthesis terminating where ddNTP
has been randomly incorporated.
The basic steps in sequencing a

small hypothetical fragment by
the Sanger-Coulson (dideoxy)
technique
DNA Sequencing
Techniques developed in the
1970s are widely used for
sequencing nucleic acids.
The Sanger-Coulson dideoxy
method became the most widely
used:
DNA fragments are
separated by gel
electrophoresis and the
DNA sequence is read from
the gel bands.
The amino acid sequence of
the protein is deduced from
the nucleotide sequence.
Gel lanes in which

fluorescently labeled
daughter molecules have
been separated. The color
of the band is determined
by the identity of the
dideoxynucleotide at the
3 end of the DNA strand
Nucleotides in the
template strand is
interpreted by a computer
that reads from the
bottom to the top of the
gel to generate an
electropherogram
DNA Sequencing
Newer techniques
Next-generation sequencing (NGS) is based on
polymerase-dependent DNA synthesis but does not
depend upon premature termination.
NGS does not require separation of strands by electrophoresis.
It identifies the individual nucleotides as they are being
incorporated by the polymerase in real time.
Third-generation (or single molecule) sequencing

Does not require amplified DNA, nor do they involve enzymatic
activities.
Accomplishes sequencing by pulling each DNA molecule
through a tiny hole, or nanopore and identifying each
nucleotide one at a time.
(18.15) DNA Libraries

DNA libraries are often produced from DNA cloning:
Genomic libraries are produced from DNA extracted from nuclei
and contain all DNA sequences of the species.
DNA fragments for the library can be obtained by cutting
genomic DNA with restriction enzymes.
Cleaving of genomic DNA is random, which generates
overlapping fragments.
Overlapping fragments are useful for chromosome walking, to
study linked sequences in an extended region of a
chromosome.
Cloning Larger DNA Fragments in Specialized Cloning Vectors

A yeast artificial chromosome (YAC) can accommodate large
(up to 1000 kb) DNA inserts.
A bacterial artificial chromosome (BAC) accepts DNA inserts
of up to 500 kb, and can be quickly grown to large numbers.
DNA Libraries
cDNA libraries
DNA libraries are often
produced from DNA
cloning:
cDNA libraries are derived
from DNA copies of an RNA
population.
The isolation of genomic
fragments allows study of
the genome.
Coding regions of a gene
can be studied using cDNAs,
synthesized by reverse
transcriptase using mRNA
as a template.
Allow foreign DNA to be
transcribed and translated
during the infection process.
Synthesizing cDNAs for cloning in a

plasmid: the generation of doublestranded DNA from mRNA (left), and
cloning the cDNA into a plasmid (right).
(18.16) DNA Transfer into

Eukaryotic Cells and Mammalian
Embryos
DNA incorporation into the

genome of a non-replicating
virus is called transduction.
DNA introduced into cultured
cells is called transfection.
The gene whose role is being
investigated after transfection is
called a transgene.
A direct way to introduce foreign
genes into a cell is by
microinjection of DNA directly
into the cell nucleus.
Animals that have been
genetically engineered to that
their chromosomes have foreign
genes are called transgenic
animals.
Microinjection
of DNA into
the nucleus of
a recently
fertilized
mouse egg
Transgenic
mice: larger
mouse (left) has
the rat growth
hormone gene
regulated by the
metallothionein
promoter
DNA Transfer into Eukaryotic Cells

and Mammalian Embryos
Transgenic Plants and

Animals
Transgenic organisms
allow scientists to
determine the effects of
overexpression of a
particular DNA
sequence.
Genetic engineering can
produce animal models
used to study human
diseases.
The main goal of genetic
engineering in plants is
to improve the efficiency
of both photosynthesis
and nitrogen fixation.
Formation of transgenic
plants using the Ti
plasmid
(18.17) Determining Eukaryotic

Gene Function by Gene Elimination
This process of learning about genotypes by studying mutant
phenotypes is known as forward genetics.
The process of reverse genetics has recently been developed, which
is based on determining phenotype (i.e., function) based on the
knowledge of genotype
In Vitro Mutagenesis
Site-directed mutagenesis (SDM) allows making small changes in a
DNA sequence.
SDM is accomplished by synthesizing a DNA containing the desired
change and allowing it to hybridize to a single-stranded normal DNA.
The polymerase elongates the replicates DNA adding nucleotides
complementary to the normal DNA.
Determining Eukaryotic Gene

Function by Gene Elimination:
Knockout mice
Knockout Mice
Knockout mice are
obtained by transfecting
embryonic stem cells,
introducing them into an
embryo, and implanting
the embryo into a
female mouse.
Germ cells containing
the knockout mutation
are heterozygous, which
can be used to obtain a
homozygous mutant
phenotype.
Genes can be assessed
for their functions.
Formation of knockout mice:

step 1 relies on derivation of ES
cells from the blastocyst and
transfecting those ES cells with
a targeting
vector
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reserved.

Knockout mice
Knockout Mice
Knockout mice are
the embryo into a
female mouse.
homozygous mutant
phenotype.
Formation of knockout
mice: step 2 shows the
antibiotic selection process.
Once targets have been
identified by PCR or Southern
blot, ES cells are used for

Knockout mice
Knockout Mice
Knockout mice are
the embryo into a
female mouse.
homozygous mutant
phenotype.
Formation of knockout
mice: step 3 shows the
generation of a chimera,
derived from both ES cells
(generates brown coat color)
and the host blastocyst
2013 John Wiley & Sons, Inc. (generates
All rights reserved.
black coat color)

Knockout mice
Knockout Mice
Knockout mice are
the embryo into a
female mouse.
homozygous mutant
phenotype.
Formation of knockout mice: step

4 goes through the breeding strategy
to demonstrate germline
transmission, and how to make
heterozygous and homozygous mice

Function by Gene Elimination: RNA
interference
RNA Interference
Specific mRNAs can be
degraded in vivo by
treating with small
double-stranded siRNA
containing part of the
sequence of the target
mRNA.
Cells treated with
RNAi cannot make the
protein encoded in the
target mRNA.
Libraries containing
thousands of siRNAs
are available for the
study of gene
function.
Determining
gene function
by RNA
interference:
a gallery of
images of
cultured
Drosophila cells
incubated with
various doublestranded
siRNAs. Cells
are screened for
the presence of
abnormal
mitotic spindles
by arresting in
metaphase.

Function by Gene Elimination: RNA
interference
RNA Interference
Specific mRNAs can be
degraded in vivo by
treating with small
double-stranded siRNA
containing part of the
sequence of the target
mRNA.
Cells treated with
RNAi cannot make the
protein encoded in the
target mRNA.
Libraries containing
thousands of siRNAs
are available for the
study of gene
function.
Determining gene function by

RNA interference: (Left) An
untreated, cultured mammalian cell
at metaphase of mitosis. (Right) The
same type of cell treated by RNAi to
knockdown Aurora B kinase, a
protein involved in the metaphase
spindle checkpoint.
(18.18) The Use of Antibodies

Antibodies are highly specific proteins produced by lymphoid tissues in
response to the presence of foreign materials.
Preparation of antibodies:
A population of polyclonal antibodies can be obtained by repeated injections of a
purified antigen into an animal.
The blood of the animal serves as a source of an antiserum.
A monoclonal antibody is produced by descendants of a single antibody-producing
cell:
Antibody-producing cells do not grow and divide in culture.
Fusion of a normal antibody-producing lymphocyte and a malignant myeloma cell will create
a viable hybridoma cell that can produce large amounts of a monoclonal antibody.
The Use of Antibodies

A monoclonal antibody is
produced by descendants of a
single antibody-producing cell:
Antibody-producing cells do not
grow and divide in culture.
Fusion of a normal antibodyproducing lymphocyte and a
malignant myeloma cell will
create a viable hybridoma cell
that can produce large amounts
of a monoclonal antibody.
Formation of monoclonal antibodies

The Use of Antibodies
Antibodies can be conjugated with a fluorescent substance

that allows visualization of antigens.
In a Western blot, antibodies can be used in conjunction with
various types of fractionation procedures to identify a
particular protein (antigen) among a mixture of proteins.
Immunoprecipitation can be performed to determine proteinprotein interaction.
Immunohistochemistry/immunocytochemistry can be done to
localize proteins within a histological sample or cell.
In direct immunofluorescence, antibodies with bound
fluorescent molecules bind to antigens and can be visualized
with a fluorescence microscope.
In indirect immunofluorescence, cells are incubated with
unlabeled antibodies, and then with labeled 2 antibody
against the 1 antibody.
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CH 18

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CH 18

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CHAPTER 18

Methods in Cell Biology

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Describe types of information that can be obtained by using various techniques.

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(18.1) The Light Microscope

The light microscope uses the refraction of light rays to

The Light Microscope

Relationship between the

Resolution is the ability to see two nearby points as distinct images.

The Light Microscope

Feulgen stain: specific for

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The Light Microscope

Feulgen stain: specific for

The Light Microscope

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The Light Microscope

Fluorescence Microscopy (and Related Fluorescence-Based

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The Light Microscope

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The Light Microscope

The Light Microscope

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The Light Microscope

Confocal fluorescence micrographs

The Light Microscope

Breaking the light microscopes limit of

Super-Resolution Fluorescence Microscopy

STORM (stochastic optical reconstruction microscopy) allows the

(18.2) Transmission Electron

Comparison of skeletal muscle

Transmission Electron Microscope

A comparison of the lens

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Transmission Electron Microscopy

Preparation of a specimen for

Transmission Electron Microscopy

The procedure used for

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Transmission Electron Microscopy

Transmission Electron Microscopy

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(18.3) Scanning Electron Atomic

SEMs: (left) T4 bacteriophage (X

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Scanning Electron Atomic Force

Atomic Force Microscopy (AFM)

(18.4) The Use of Radioisotopes

Radioisotopes can be easily

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The Use of Radioisotopes

The Use of Radioisotopes

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(18.5) Cell Culture

Cells can be obtained in large quantities.

Cells in a culture require media that includes hormones

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Labs are moving to three-dimensional cultures in which cells

(18.6) The Fractionation of a Cells