Lecture PowerPoint to accompany
Molecular Biology
Fifth Edition
Robert F. Weaver
Chapter 5
Molecular Tools for
Studying Genes and
Gene Activity
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5.1 Molecular Separations
• Often mixtures of proteins or nucleic acids
are generated during the course of
molecular biological procedures
– A protein may need to be purified from a
crude cellular extract
– A particular nucleic acid molecule made in a
reaction needs to be purified
• Gel electrophoresis is used to separate
different species of:
– Nucleic acid
– Protein
5-2
DNA Gel Electrophoresis
• Melted agarose is poured into
a form equipped with
removable comb
• Comb “teeth” form slots in the
solidified agarose
• DNA samples are placed in
the slots
• An electric current is run
through the gel at a neutral
pH to allow the sample to
travel through the gel matrix
5-3
DNA Separation by Agarose Gel
Electrophoresis
• DNA is negatively charged due to the
phosphates in its backbone and moves
toward the positive pole
– Small DNA pieces have little frictional
drag so they move rapidly
– Large DNAs have more frictional drag
so their mobility is slower
– Distributes DNA according to size
• Largest near the top
• Smallest near the bottom
• DNA is stained with fluorescent dye that
intercalates between the bases
5-4
DNA Size Estimation
• Mobility of fragments are
plotted v. log of molecular
weight (or number of base pairs)
• Electrophoresis of unknown
DNA in parallel with standard
fragments permits size
estimation upon comparison
• Same principles apply to
RNA separation
5-5
Electrophoresis of Large DNA
• Special techniques are required for DNA
fragments larger than about 1 kilobases
• Instead of constant current, alternate long
pulses of current in forward direction with
shorter pulses in either opposite or
sideways direction
• Technique is called pulsed-field gel
electrophoresis (PFGE)
5-6
Protein Gel Electrophoresis
• Separation of proteins is done using
polyacrylamide gel electrophoresis (PAGE)
– Treat proteins to denature subunits with
detergent such as sodium dodecyl sulfate
(SDS)
• SDS coats polypeptides with negative charges so all
move to anode
• Masks natural charges of protein subunits so all
move relative to mass not charge
– As with DNA smaller proteins move faster
toward the anode
5-7
Summary
• DNAs, RNAs, and proteins of various
masses can be separated by gel
electrophoresis
• Most common gel used in nucleic acid
electrophoresis is agarose but
polyacrylamide is typically used in protein
electrophoresis
• SDS-PAGE is used to separate
polypeptides according to their masses
5-8
Two-Dimensional Gel Electrophoresis
• While SDS-PAGE gives good resolution of
polypeptides, some mixtures are so
complex that additional resolution is
needed
• Two-dimensional gel electrophoresis:
– Nondenaturing gel electrophoresis (no SDS)
uses 2 consecutive gels each in a different
dimension
– Sequential gels with distinct pH separation
and polyacrylamide gel concentration
5-9
A Simple 2-D Method
• Samples are run in 2 gels
– First dimension separates using one
concentration of polyacrylamide at one pH
– Second dimension uses different
concentration of polyacrylamide and pH
– Proteins move differently at different pH
values without SDS and at different
acrylamide concentrations
5-10
A More Powerful Two-Dimensional Gel
Electrophoresis Technique
A two process method:
• Isoelectric focusing gel: mixture of proteins
electrophoresed through gel in a narrow
tube containing a pH gradient
– Negatively charged protein moves to its
isoelectric point at which it is no longer
charged
– Tube gel is removed and used as the sample
in the second process
5-11
A More Powerful Two-Dimensional Gel
Electrophoresis Technique continued
• Standard SDS-PAGE:
– Tube gel is removed and used as the sample
at the top of a standard polyacrylamide gel
– Proteins partially resolved by isoelectric
focusing are further resolved according to size
• When used to a compare complex mixtures
of proteins prepared under two different
conditions, even subtle differences are
visible
5-12
Ion-Exchange Chromatography
• Chromatography originally referred to the
pattern seen after separating colored
substances on paper
• Ion-exchange chromatography uses a
resin to separate substances by charge
• This is especially useful for proteins
• Resin is placed in a column and the
sample is loaded onto the column material
5-13
Separation by Ion-Exchange
Chromatography
• Once the sample is
loaded buffer is passed
over the resin + sample
• As ionic strength of
elution buffer increases,
samples of solution
flowing through the
column are collected
• Samples are tested for
the presence of the
protein of interest
5-14
Gel Filtration Chromatography
• Protein size is a valuable property that can be
used as a basis of physical separation
• Gel filtration uses columns filled with porous
resins that let in smaller substances and exclude
larger substances
• As a result larger substances travel faster
through the column
5-15
Affinity Chromatography
• The resin contains a substance to which the
molecule of interest has a strong and
specific affinity
• The molecule binds to a column resin
coupled to the affinity reagent
– Molecule of interest is retained
– Most other molecules flow through without
binding
– Last, the molecule of interest is eluted from the
column using a specific solution that disrupts
their specific binding
5-16
Summary
• High-resolution separation of proteins can be
achieved by two-dimensional gel electrophoresis
• Ion-exchange chromatography can be used to
separate substances according to their sizes
• Gel filtration chromatography uses columns filled
with porous resins that let in smaller substances
but exclude larger ones
• Affinity chromatography is a powerful purification
technique that exploits an affinity reagent with
strong and specific affinity for a molecule of
interest
5-17
5.2 Labeled Tracers
• For many years “labeled” has been
synonymous with “radioactive”
• Radioactive tracers allow vanishingly small
quantities of substances to be detected
• Molecular biology experiments typically
require detection of extremely small
amounts of a particular substance
5-18
Autoradiography
Autoradiography is a means of
detecting radioactive
compounds with a
photographic emulsion
– Preferred emulsion is x-ray film
– DNA is separated on a gel and
radiolabeled
– Gel is placed in contact with xray film for hours or days
– Radioactive emissions from the
labeled DNA expose the film
– Developed film shows dark
bands
5-19
Autoradiography Analysis
• Relative quantity of
radioactivity can be assessed
looking at the developed film
• More precise measurements
are made using a densitometer
– Area under peaks on a tracing by
a scanner
– Proportional to darkness of the
bands on autoradiogram
5-20
Phosphorimaging
This technique is more accurate in quantifying
amount of radioactivity in a substance
– Response to radioactivity is much more linear
– Place gel with radioactive bands in contact with
a phosphorimager plate
– Plate absorbs b electrons that excite molecules
on the plate which remain excited until plate is
scanned
– Molecular excitation is monitored by a detector
5-21
Liquid Scintillation Counting
Radioactive emissions from a sample create
photons of visible light are detected by a
photomultiplier tube in the process of liquid
scintillation counting
– Remove the radioactive material (band from gel) to a
vial containing scintillation fluid
– Fluid contains a fluor that fluoresces when hit with
radioactive emissions
– Acts to convert invisible radioactivity into visible light
5-22
Nonradioactive Tracers
• Newer nonradioactive tracers now rival
older radioactive tracers in sensitivity
• These tracers do not have hazards:
– Health exposure
– Handling
– Disposal
• Increased sensitivity is from use of a
multiplier effect of an enzyme that is
coupled to probe for molecule of interest
5-23
Detecting Nucleic Acids With a
Nonradioactive Probe
5-24
5.3 Using Nucleic Acid Hybridization
• Hybridization is the ability of one singlestranded nucleic acid to form a double
helix with another single strand of
complementary base sequence
• Previous discussion focused on colony
and plaque hybridization
• This section looks at techniques for
isolated nucleic acids
5-25
Southern Blots: Identifying Specific
DNA Fragments
• Digests of genomic DNA are separated on a gel
• The separated pieces are transferred to filter
(nitrocellulose) by diffusion, or more recently by
electrophoresing the DNA onto the filter
• The filter is then treated with alkali to denature the
DNA, resulting ssDNA binds to the filter
• A labeled cDNA probe that is complementary to the
DNA of interest is then applied to the filter
• A positive band should be detectable where
hybridization between the probe and DNA occurred
5-26
Southern Blots
• The probe hybridizes and a
band is generated
corresponding to the DNA
fragment of interest
• Visualize bands with x-ray
film or autoradiography
• Multiple bands can lead to
several interpretations
– Multiple genes
– Several restriction sites in the
gene
5-27
DNA Fingerprinting and DNA Typing
• Southern blots are used in forensic labs to
identify individuals from DNA-containing
materials
• Minisatellite DNA is a sequence of bases
repeated several times, also called a DNA
fingerprint
– Individuals differ in the pattern of repeats of
the basic sequence
– The difference is large enough that 2 people
have only a remote chance of having exactly
the same pattern of repeats
5-28
DNA Fingerprinting
Process is a Southern blot
• Cut the DNA under study
with restriction enzyme
– Ideally cut on either side of
minisatellite but not inside
• Run the digested DNA on a
gel and blot
• Probe with labeled
minisatellite DNA and image
– Note that real samples result in
very complex patterns
5-29
Forensic Uses of DNA Fingerprinting
and DNA Typing
• While people have different DNA fingerprints,
parts of the pattern are inherited in a Mendelian
fashion
– Can be used to establish parentage
– Potential to identify criminals
– Remove innocent people from suspicion
• Actual pattern has so many bands they can
smear together indistinguishably
– Forensics uses probes for just a single locus
– Set of probes gives a set of simple patterns
5-30
In Situ Hybridization: Locating Genes in
Chromosomes
• Labeled probes can be used to hybridize to
chromosomes and reveal which chromosome
contains the gene of interest
– Spread chromosomes from a cell
– Partially denature DNA creating single-stranded
regions to hybridize to labeled probe
– Stain chromosomes and detect presence of label on
particular chromosome
• Probe can be detected with a fluorescent
antibody in a technique called fluorescence in
situ hybridization (FISH)
5-31
Immunoblots
Immunoblots (also called Western blots) use a
similar process to Southern blots
– Electrophoresis of proteins
– Blot the proteins from the gel to a membrane
– Detect the protein using antibody or antiserum to
the target protein
– Labeled secondary antibody is used to bind the
first antibody for visualization and to increase
the signal
5-32
Summary
• Labeled DNA (or RNA) probes can be used to
hybridize to DNAs of the same or very similar
sequence on a Southern blot
• DNA fingerprinting can be used as a forensic tool
or to test parentage
• In situ hybridization can be used to locate genes
or other specific DNA sequences on whole
chromosomes
• Proteins can be detected and quantified in a
complex mixture using Western blots
5-33
5.4 DNA Sequencing
• Sanger, Maxam, and Gilbert developed 2 methods
for determining the exact base sequence of a
cloned piece of DNA
• Modern DNA sequencing is based on the Sanger
method and uses dideoxy nucleotides to terminate
DNA synthesis
– The process yields a series of DNA fragments whose
size is measured by electrophoresis
– The last base in each fragment is known as that dideoxy
nucleotide was used to terminate the reaction
– Ordering the fragments by size tells the base sequence
of the DNA
5-34
Sanger Method of DNA Sequencing
5-35
Automated DNA Sequencing
• Manual sequencing is
powerful but slow
• Automated sequencing
uses dideoxynucleotides
tagged with different
fluorescent molecules
– Products of each
dideoxynucleotide will
fluoresce a different color
– Four reactions are
completed, then mixed
together and run out on one
lane of a gel
5-36
High Throughput Sequencing
• Once an organism’s genome sequence is known,
very rapid sequencing techniques can be applied
to sequence the genome of another member of the
same species
• Produces relatively short reads or contiguous
sequences (25-35bp or 200-300bp, depending on
the method) that can easily be pieced together if a
reference sequence is available
5-37
High Throughput Sequencing
• Pyrosequencing is one example that is an automated
system with the advantages of speed and accuracy
- nucleotides are added one by one and the incorporation of
a nucleotide is detected by a release of pyrophosphate,
which leads to a flash of light
• Another method (Illumina company) starts by attaching
short pieces of DNA to a solid surface, amplifying each
DNA in a tiny patch on the surface, then sequencing the
patches together by extending them one nucleotide at a
time using fluorescent chain-terminating nucleotides, whose
fluoresce reveals their identity
5-38
Restriction Mapping
• Prior to the start of large-scale sequencing
preliminary work is done to locate
landmarks
– A map based on physical characteristics is
called a physical map
– If restriction sites are the only map features
then a restriction map has been prepared
5-39
Restriction Map Example
• Consider a 1.6 kb piece of
DNA as an example
• Cut separate samples of the
original 1.6 kb fragment with
different restriction enzymes
• Separate the digests on an
agarose gel to determine the
size of pieces from each
digest
• Can also use same digest to
find the orientation of an insert
5-40
cloned into a vector
Southern Blots and Restriction Mapping
5-41
Summary
• Physical maps tell about the spatial arrangement
of physical “landmarks” such as restriction sites
– In restriction mapping cut the DNA in question with 2
or more restriction enzymes in separate reactions
– Measure the sizes of the resulting fragments
– Cut each with another restriction enzyme and
measure size of subfragments by gel electrophoresis
• Sizes permit location of some restriction sites
relative to others
• Improve process by Southern blotting fragments
and hybridizing them to labeled fragments from
another restriction enzyme to reveal overlaps 5-42
5.5 Protein Engineering With Cloned
Genes: Site-Directed Mutagenesis
• Cloned genes permit biochemical
microsurgery on proteins
– Specific bases in a gene may be changed
– Amino acids at specific sites in the protein
product may be altered as a result
– Effects of those changes on protein function
can be observed
5-43
PCR-based Site-Directed Mutagenesis
5-44
Summary
• Using cloned genes, one can introduce changes
that may alter the amino acid sequence of the
corresponding protein products
• Mutagenized DNA can be made with:
– Double-stranded DNA
– Two complementary mutagenic primers
– PCR
• Digest the PCR product to remove wild-type
DNA
• Cells can be transformed with mutagenized DNA
5-45
5.6 Mapping and Quantifying Transcripts
• In the field of molecular biology mapping
(locating start and end) and quantifying
(how much transcript exists at a set time)
transcripts are common procedures
• Often transcripts do not have a uniform
terminator, resulting in a continuum of
species smeared on a gel
• Techniques that are specific for the
sequence of interest are important
5-46
Northern Blots
• Northern blots detect RNA
• Example: You have cloned a cDNA
– Question: How actively is the corresponding
gene expressed in different tissues?
– Answer: Find out using a Northern Blot
• Obtain RNA from different tissues
• Run RNA on agarose gel and blot to membrane
• Hybridize to a labeled cDNA probe
– Northern plot tells abundance of the transcript
– Quantify using densitometer
5-47
S1 Mapping
Use S1 mapping to locate the ends of RNAs
and to determine the amount of a given RNA
in cells at a given time
– Label a ssDNA probe that can only hybridize
to transcript of interest
– Probe must span the sequence start to finish
– After hybridization, treat with S1 nuclease
which degrades ssDNA and RNA
– Transcript protects part of the probe from
degradation
– Size of protected area can be measured by
gel electrophoresis
5-48
S1 Mapping the 5’ End
5-49
S1 Mapping the 3’ End
5-50
Summary
• A Northern blot is similar to a Southern blot
but is a method used for detection of RNA
• In S1 mapping, a labeled DNA probe is used
to detect 5’- or 3’-end of a transcript
• Amount of probe protected is proportional to
concentration of transcript, so S1 mapping
can be quantitative
• RNase mapping is a variation on SI mapping
that uses an RNA probe and RNase
5-51
Primer Extension Schematic
Primer extension works to determine the 5’-end of a
transcript to one-nucleotide accuracy
• Start with in vivo transcription,
harvest cellular RNA containing
desired transcript
• Hybridize labeled
oligonucleotide [18nt] (primer)
• Reverse transcriptase extends
the primer to the 5’-end of
transcript
• Denature the RNA-DNA hybrid
and run the mix on a highresolution DNA gel
• Can estimate transcript
concentration also
5-52
Run-Off Transcription
• A good assay to measure the
rate of in vitro transcription
• DNA fragment containing
gene to transcribe is cut with
restriction enzyme in middle of
transcription region
• Transcribe the truncated
fragment in vitro using labeled
nucleotides, as polymerase
reaches truncation it “runs off”
the end
• Measure length of run-off
transcript compared to
location of restriction site at 3’end of truncated gene
5-53
Schematic of the G-Less Cassette Assay
A variation of the run-off technique in which a stretch of
nucleotides lacking guanines is inserted into the nontemplate
strand just downstream of the promoter
• Transcribe altered template
in vitro with CTP, ATP and
UTP one of which is labeled,
but no GTP
• Transcription will stop when
the first G is required
resulting in an aborted
transcript of predictable size
• Separate transcripts on a
gel and measure
transcription activity with
autoradiography
5-54
Summary
• Run-off transcription is a means of checking
efficiency and accuracy of in vitro transcription
– Gene is truncated in the middle and transcribed in vitro in
presence of labeled nucleotides
– RNA polymerase runs off the end making an incomplete
transcript
– Size of run-off transcript locates transcription start site
– Amount of transcript reflects efficiency of transcription
• In G-less cassette transcription, a promoter is fused
to dsDNA cassette lacking Gs in nontemplate strand
– Construct is transcribed in vitro in absence of of GTP
– Transcription aborts at end of cassette for a predictable
size band on a gel
5-55
5.7 Measuring Transcription Rates in Vivo
• Primer extension, S1 mapping and
Northern blotting will determine the
concentration of specific transcripts at a
given time
• These techniques do not really reveal the
rate of transcript synthesis as
concentration involves both:
– Transcript synthesis
– Transcript degradation
5-56
Nuclear Run-On Transcription
• The idea of this assay is to isolate nuclei
from cells, allow them to extend in vitro the
transcripts already started in vivo
• RNA polymerase that has already initiated
transcription will “run-on” or continue to
elongate the same RNA chains
• Effective as initiation of new RNA chains in
isolated nuclei does not generally occur
• Results will show transcription rates and an
idea of which genes are transcribed
5-57
Nuclear Run-On Transcription Diagram
5-58
Reporter Gene Transcription
• Place a surrogate reporter gene under the
control of a specific promoter and measure the
accumulation of the product of this reporter gene
• The reporter genes are carefully chosen to have
products very convenient to assay
– lacZ produces b-galactosidase which has a blue
cleavage product
– cat produces chloramphenicol acetyl transferase
(CAT) which inhibits bacterial growth
– Luciferase produces a chemiluminescent compound
that emits light
5-59
Measuring Protein Accumulation in Vivo
• Gene activity can be monitored by measuring the
accumulation of protein, the ultimate gene product
• There are two primary methods of measuring
protein accumulation
– Immunoblotting / Western blotting (discussed earlier)
– Immunoprecipitation
• Immunoprecipitation typically uses an antibody
that will bind specifically to the protein of interest
followed with a secondary antibody complexed to
Protein A on resin beads using a low-speed
centrifuge
5-60
5.8 Assaying DNA-Protein Interactions
• Study of DNA-protein interactions is of
significant interest to molecular biologists
• Types of interactions often studied:
– Protein-DNA binding
– Which bases of DNA interact with a protein
5-61
Filter Binding
Filter binding is used to measure DNAprotein interaction and based on the fact that
double-stranded DNA will not bind by itself
to a filter, but a protein-DNA complex will
– Double-stranded DNA can be labeled and
mixed with protein
– Assay protein-DNA binding by measuring the
amount of label retained on the filter
5-62
Nitrocellulose Filter-Binding Assay
• dsDNA is labeled and mixed with protein
• Pour dsDNA through a nitrocellulose filter
• Measure amount of radioactivity that passed
through filter and retained on filter
5-63
Gel Mobility Shift
• DNA moves through a gel faster when it is not
bound to protein
• Gel shift assays detect interaction between
protein and DNA by reduction of the
electrophoretic mobility of a small DNA bound to
a protein
5-64
Footprinting
• Footprinting detects protein-DNA
interaction and will show where a target
lies on DNA and which bases are involved
in protein binding
• Three methods are very popular:
– DNase footprinting
– Dimethylsulfate footprinting
– Hydroxyl radical footprinting
5-65
DNase Footprinting
Protein binding to DNA covers
the binding site and protects from
attack by DNase
• End label DNA, 1 strand only
• Protein binds DNA
• Treat complex with DNase I
mild conditions for average
of 1 cut per molecule
• Remove protein from DNA,
separate strands and run on
a high-resolution
polyacrylamide gel
5-66
DMS Footprinting
• Starts the same way as
DNase footprinting but then
methylate with DMS at
conditions for 1 methylation
per DNA molecule
• The protein is then dislodged
and treated to remove the
methylated purines resulting
in apurinic sites which breaks
the DNA
• The DNA fragments are then
electrophoresed and
autoradiograped for detection
5-67
Summary
• Footprinting finds target DNA sequence or
binding site of a DNA-binding protein
• DNase footprinting binds protein to end-labeled
DNA target, then attacks DNA-protein complex
with DNase
• DNA fragments are electrophoresed with protein
binding site appearing as a gap in the pattern
where protein protected DNA from degradation
• DMS, DNA methylating agent is used to attack
the DNA-protein complex
• Hydroxyl radicals – copper- or iron-containing
organometallic complexes generate hydroxyl
5-68
radicals that break the DNA strands
Chromatin Immunoprecipitation (ChIP)
• ChIP is a method used to discover whether a
given protein is bound to a given gene in
chromatin - the DNA-protein complex that is the
natural state of the DNAnin a living cell
• ChIP uses an antibody to precipitate a particular
protein in complex with DNA, and PCR to
determine whether the protein binds near a
particular gene
5-69
Chromatin Immunoprecipitation (ChIP)
5-70
5.9 Assaying Protein-Protein Interactions
• Immunoprecipitation uses an antibody that will
bind specifically to the protein of interest and,
using a low-speed centrifuge, will ‘pull-down’ any
proteins associated with the protein of interest
• The yeast-two-hybrid assay is used to
demonstrate binding (even transient) between
two proteins
• The yeast-two-hybrid assay can also be used to
fish for unknown proteins that interact with a
known protein
5-71
The Yeast-Two Hybrid Assay
5-72
5.10 Finding RNA Sequences That
Interact With Other Molecules
• SELEX is systematic evolution of ligands by
exponential enrichment
• SELEX is a method to find RNA sequences that
interact with other molecules, even proteins
– RNAs that interact with a target molecule are selected
by affinity chromatography
– Convert to dsDNA and amplify by PCR
– RNAs are now highly enriched for sequences that
bind to the target molecule
5-73
Functional SELEX
• Functional SELEX is a variation where the
desired function alters RNA so it can be
amplified
• If desired function is enzymatic,
mutagenesis can be introduced into the
amplification step to produce variants with
higher activity
5-74
5.11 Knockouts and Transgenes
• Probing structures and activities of genes does
not answer questions about the role of the gene
in the life of the organism
• Targeted disruption of genes is now possible in
several organisms
• When genes are disrupted in mice the products
are called knockout mice
• Foreign genes, called transgenes, can also be
added to an organism, such as a mouse, to
create transgenic mice
5-75
Stage 1 of the Knockout Mouse
• Cloned DNA containing the mouse gene to be
knocked out is interrupted with another gene that
confers resistance to neomycin
• A thymidine kinase gene is placed outside the target
gene
• Mix engineered mouse DNA with stem cells so
interrupted gene will find way into nucleus and
homologous recombination will occur between the
altered gene and the resident, intact gene
• These events are rare, many cells will need to be
screened using the introduced genes
5-76
Making a Knockout Mouse: Stage 1
5-77
Stage 2 of the Knockout Mouse
• Introduce the interrupted gene into a whole
mouse
• Inject engineered cells into a mouse blastocyst
• Implant the embryo into a surrogate mother who
will give birth to chimeric mouse
• True heterozygote results when chimera mates
with a black mouse to produce brown mice, half
of which will have interrupted gene
5-78
Making a Knockout Mouse: Stage 2
5-79
Knockout Results
• Phenotype may not be obvious in the
progeny, but still instructive
• Other cases can be lethal with the mice
dying before birth
• Intermediate effects are also common and
may require monitoring during the life of
the mouse
5-80
Methods to Generate Transgenic Mice
• Two methods to generate transgenic mice:
• 1. Injection of cloned foreign gene into the sperm
pronucleus just after fertilization of a mouse egg
but before the sperm and egg nuclei have fused
to allow for insertion of the foreign DNA into the
embryonic cell DNA
• 2. Injection of cloned foreign DNA into mosue
embryonic stem cells, creating transgenic ES
cells
• Both methods produce chimeric mice that must
undergo several rounds of breeding and
selection to find true transgenic animals
5-81
Summary
• To probe the role of a gene, molecular
biologists can perform targeted disruption
of the corresponding gene in a mouse and
then look for the effects of the mutation in
the ‘knockout mouse’ or insert the foreign
gene as a transgene in the ‘transgenic
mouse’
5-82