Transcript Inquiry into Life Twelfth Edition

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 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
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 b-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