Lecture PowerPoint to accompany

Molecular Biology
Fourth 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

5-2

Gel Electrophoresis
Gel electrophoresis is used to separate
different species of:
Nucleic acid
Protein

5-3

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
5-4

DNA Separation by Agarose Gel

Electrophoresis
DNA is negatively charged due to
phosphates in its backbone and
moves to anode, the positive pole
Small DNA pieces have little
frictional drag so move rapidly
Large DNAs have more frictional
drag so their mobility is slower
Result distributes DNA according
to size
Largest near the top
Smallest near the bottom

DNA is stained with fluorescent

dye

5-5

DNA Size Estimation

Comparison with standards
permits 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
Same principles apply to RNA
separation
5-6

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)
5-7

Protein Gel Electrophoresis

Separation of proteins is done using a gel
made of polyacrylamide (polyacrylamide
gel electrophoresis = PAGE)
Treat proteins to denature subunits with
detergent such as 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

5-8

Summary
DNAs, RNAs, and proteins of various
masses can be separated by gel
electrophoresis
Most common gel used in nucleic acid
electrophoresis is agarose
Polyacrylamide is usually used in protein
electrophoresis
SDS-PAGE is used to separate
polypeptides according to their masses
5-9

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 can
be done:
(no SDS) uses 2 consecutive gels
Sequential gels with first a pH separation,
then separate in a polyacrylamide gel
5-10

A Simple 2-D Method

Run samples 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

5-11

Two-Dimensional Gel
Electrophoresis Details
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
5-12

More Two-Dimensional Gel

Electrophoresis Details
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
5-13

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 sample
loaded onto the column material
5-14

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
5-15

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, exclude larger ones
Larger substances travel faster through the column

5-16

Affinity Chromatography
In 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 the
specific binding
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 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 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
agarose gel
The separated pieces are transferred to
filter by diffusion, or more recently by
electrophoresing the bands onto the filter
Filter is treated with alkali to denature the
DNA, resulting ssDNA binds to the filter
Probe the filter using labeled cDNA
5-26

Southern Blots
Probe cDNA 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 DNA
fingerprint
Individuals differ in the pattern of repeats of
the basic sequence
Difference is large enough that 2 people have
only a remote chance of having exactly the
same pattern
5-28

DNA Fingerprinting
Process really just a Southern
blot
Cut the DNA under study
with restriction enzyme
Ideally cut on either side of
minisatellite but not inside

Run digest on a gel and blot

Probe with labeled
minisatellite DNA and imaged
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 and increase the signal
5-32

Western Blots

5-33

DNA Sequencing
Sanger, Maxam, 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

5-34

Sanger Manual Sequencing

Sanger DNA sequencing method uses
dideoxy nucleotides to terminate DNA
synthesis
The process yields a series of DNA fragments
whose size is measured by electrophoresis
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
5-35

Sanger DNA Sequencing

5-36

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
5-37

Automated DNA Sequencing

5-38

Restriction Mapping
Prior to 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

Consider a 1.6 kb piece of DNA as an

example
5-39

Restriction Map 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 cloned into a vector
5-40

Mapping Experiment

5-41

Using Restriction Mapping With

an Unknown DNA Sample

5-42

Mapping the Unknown

5-43

Southern Blots and Restriction

Mapping

5-44

Summary
Physical map tells 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
5-45

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 also be altered
Effects of those changes on protein function can be
observed

Might investigate the role of phenolic group on

tyrosine compared to phenylalanine
5-46

Site-Directed Mutagenesis With

PCR

5-47

Summary
Using cloned genes, can introduce changes at
will to alter amino acid sequence of 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
5-48

5.4 Mapping and Quantifying

Transcripts
Mapping (locating start and end) and
quantifying (how much transcript exists at
a set time) are common procedures
Often transcripts do not have a uniform
terminator, resulting in a continuum of
species smeared on a gel
Techniques that specific for the sequence
of interest are important
5-49

Northern Blots
You have cloned a cDNA
How actively is the corresponding gene
expressed in different tissues?
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
5-50

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
5-51

S1 Mapping the 5 End

5-52

S1 Mapping the 3 End

5-53

Summary
In S1 mapping, a labeled DNA probe is used to
detect 5- or 3-end of a transcript
Hybridization of the probe to the transcript protects
a portion of the probe from digestion by S1
nuclease, specific for single-stranded
polynucleotides
Length of the section of probe protected by the
transcript locates the end of the transcript relative
to the known location of an end of the probe
Amount of probe protected is proportional to
concentration of transcript, so S1 mapping can be
quantitative
RNase mapping uses an RNA probe and RNase
5-54

Primer Extension
Primer extension works to determine exactly
the 5-end of a transcript to one-nucleotide
accuracy
Specificity of this method is due to
complementarity between primer and transcript

S1 mapping will give similar results but limits:

S1 will nibble ends of RNA-DNA hybrid
Also can nibble A-T rich regions that have
melted
Might not completely digest single-stranded
regions

5-55

Primer Extension Schematic

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 high-resolution DNA gel
Can estimate transcript
concentration also

5-56

Run-Off Transcription and GLess Cassette Transcription

If want to assess:
Transcription accuracy
How much of this accurate transcription

Simpler method is run-off transcription

Can be used after the physiological start
site is found by S1 mapping or primer
extension
Useful to see effects of promoter mutation
on accuracy and efficiency of transcription
5-57

Run-Off 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
5-58

G-Less Cassette Assay

Variation of the run-off technique, instead
of cutting the gene with restriction
enzyme, insert a stretch of nucleotides
lacking guanines in nontemplate strand
just downstream of promoter
As promoter is stronger a greater number
of aborted transcripts is produced

5-59

Schematic of the G-Less

Cassette Assay
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

5-60

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
5-61

5.5 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
5-62

Nuclear Run-On Transcription

Isolate nuclei from cells, allow them to
extend in vitro the transcripts already
started in vivo in a technique called run-on
transcription
RNA polymerase that has already initiated
transcription will run-on or continue to
elongate same RNA chains
Effective as initiation of new RNA chains in
isolated nuclei does not generally occur
5-63

Run-On Analysis
Results will show transcription rates and
an idea of which genes are transcribed
Identification of labeled run-on transcripts
is best done by dot blotting
Spot denatured DNAs on a filter
Hybridize to labeled run-on RNA
Identify the RNA by DNA to which it hybridizes

Conditions of run-on reaction can be

manipulated with effects of product can be
measured
5-64

Nuclear Run-On Transcription

Diagram

5-65

Reporter Gene Transcription

Place a surrogate reporter gene under control of
a specific promoter, measure accumulation of
product of this reporter gene
Reporter genes are carefully chosen to have
products very convenient to assay
lacZ produces -galactosidase which has a blue
cleavage product
cat produces chloramphenicol acetyl transferase
(CAT) which inhibits bacterial growth
Luciferase produces chemiluminescent compound
that emits light
5-66

Measuring Protein Accumulation

in Vivo
Gene activity can be monitored by
measuring the accumulation of protein
(the ultimate gene product)
Two primary methods of measuring
protein accumulation
Immunoblotting / Western blotting
Immunoprecipitation

5-67

Immunoprecipitation
Label proteins by growing cells with 35Slabeled amino acid
Bind protein of interest to an antibody
Precipitate the protein-antibody complex
with a secondary antibody complexed to
Protein A on resin beads using a lowspeed centrifuge
Determine protein level with liquid
scintillation counting
5-68

5.6 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
5-69

Filter Binding
Filter binding to measure DNA-protein
interaction is based on the fact that doublestranded 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
5-70

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

5-71

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

5-72

Footprinting
Footprinting shows 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

5-73

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

5-74

DMS Footprinting
Dimethylsulfate
(DMS) is a
methylating agent
which can fit into DNA
nooks and crannies
Starts as DNase, then
methylate with DMS
at conditions for 1
methylation per DNA
molecule
5-75

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
radicals that break the DNA strands
5-76

5.7 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

5-77

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

5-78

5.8 Knockouts
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

5-79

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 with altered gene and
resident, intact gene
These events are rare, many cells will need to be
screened using the introduced genes
5-80

Making a Knockout Mouse:

Stage 1

5-81

Stage 2 of the Knockout Mouse

Introduce the interrupted gene into a whole
mouse
Inject engineered cells into a mouse blastocyst
Embryo into a surrogate mother who gives birth
to chimeric mouse with patchy coat
True heterozygote results when chimera mates
with a black mouse to produce brown mice, half
of which will have interrupted gene

5-82

Making a Knockout Mousse:

Stage 2

5-83

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

5-84