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Chapt. 5 Molecular Tools for Studying
Genes and Gene Activity
Learning outcomes:
• Be able to state basic principles
and processes of selected tools
• Demonstrate ability to interpret
experiments with these tools
• Important Figs: 2, 4, 6, 7, 8, 11*, 12,
15, 17, 18*, 20, 26
• Review Q: 1, 2, 4, 5, 7, 8, 9, 10, 11,
12, 21; Analyt Q: 1, 2
5-1
5.1 Molecular Separations
Mixtures of proteins or nucleic acids made during
molecular biological procedures
– A protein purified from crude cellular extract
– A particular nucleic acid molecule made in a
reaction needs to be purified
Gel electrophoresis and chromatography
widely used techniques for separating specific
molecules from mixture
5-2
DNA Gel Electrophoresis
• Melted agarose poured into
mold with comb
• Comb “teeth” form slots
(wells) in solidified agarose
• DNA samples placed in wells
• Electric current through gel
separates molecules
• Stain DNA with EtBr
• (Recall BL 261, BL 415)
Fig. 1
5-3
DNA Separation by Agarose Gel
Electrophoresis
DNA negatively charged (phosphates)
• moves to anode, positive pole
– Small DNA pieces move rapidly (little
frictional drag)
– Large DNAs move slower
– DNA distributes according to size:
• Largest near top
• Smallest near bottom
• DNA visualized by staining with
fluorescent dye (EtBr) (Fig. 2a)
+
5-4
DNA Size Estimation - Compare with standards
• Mobility of fragments plotted
v. log of molecular weight
(log number of base pairs)
– linear for smaller ones
• Electrophoresis of unknown
DNA in parallel with
standard fragments permits
size estimation
• Special techniques for huge DNA
• Similar principles apply to RNA
separation (denature)
Fig. 2b
5-5
Protein Gel Electrophoresis
-
• Separate proteins on polyacrylamide
gel (polyacrylamide gel
electrophoresis = PAGE)
– denature protein subunits with
detergent SDS
• SDS coats polypeptides with negative
charges so all move to anode
• Masks natural charges of proteins, so
all move relative to mass not charge
– Smaller proteins move faster
toward the anode; stain proteins;
compare to protein standards
+
5-6
Fig. 4
Ion-Exchange
Chromatography
Resin separates
substances by charge
• Protein sample loaded; buffer passed over resin
• Ionic strength of buffer increases, samples flowing
through column are collected
• Samples tested for presence of protein of interest
(A 280, protein gel, enzyme activity; Fig. 6)
5-7
Gel Filtration Chromatography
Gel filtration uses columns filled with porous resins:
let in smaller substances, exclude larger ones
(ex. sephadex); elute with only one buffer
Protein size - basis of physical separation
Larger substances travel faster through the column
Fig. 7
5-8
Affinity Chromatography
• Resin contains a substance to which molecule of
interest has strong and specific affinity
• Molecule binds to resin having affinity reagent
– Molecule of interest is retained
– Other molecules flow through without binding
– Molecule of interest eluted from column using
specific solution that disrupts specific binding
Ex. His-tagged proteins bind Ni+ resin; eluted with
increasing imidazole concentrations
5-9
5.2 Labeled Tracers
- often means “radioactive”
• Detect very small quantities of
proteins, DNA, RNA
• Autoradiography detects
radioactive compounds with
photographic emulsion
–
–
–
–
x-ray film
Radiolabeled DNA on gel
Gel in contact with x-ray film
Radioactive emissions from
labeled DNA expose film
– Developed film shows dark bands
5-10
Autoradiography Analysis
• Relative quantity of
radioactivity assessed from
developed film
• More precise measurements
are made with densitometer
– Area under peaks by scanner
(Fig. 9)
– Proportional to darkness of
bands on autoradiogram
5-11
Nonradioactive Tracers
• Nonradioactive tracers rival radioactive tracers in
sensitivity
• These tracers do not have hazards:
– Health exposure
– Handling
– Disposal
• Increased sensitivity because multiplier effect of
enzyme coupled to probe for molecule of interest
5-12
Detecting Nucleic Acids With Nonradioactive Probe
Biotinylated DNA probe; detect with avidin-alkaline phosphatase;
Other probes labeled with Digoxigenin; detect with antibody
coupled to alkaline phosphatase (BL 427)
Fig. 11
5-13
5.3 Using Nucleic Acid Hybridization
• Hybridization - single-stranded nucleic acid forms
double helix with another single strand of
complementary base sequence (RNA, DNA)
• Previous colony and plaque hybridization
• Detect with nucleic acid probes
• Techniques for isolated nucleic acids
5-14
Southern Blots
• Separate DNA on gel;
denature, transfer to filter
• Probe DNA hybridizes;
• Band corresponds to DNA
fragment of interest
• Visualize bands with X-ray
film, nonradioactive method
• Multiple bands lead to several
interpretations
– Multiple genes
– Several restriction sites in gene
5-15
Fig. 12
DNA Fingerprinting and DNA Typing
• Southern blots in forensic labs identify individuals
from DNA-containing materials (Jeffreys et al., 1986)
• Minisatellite DNA - sequence of bases repeated
several times, also called DNA fingerprint
– Individuals differ in repeats of basic sequence –
– Difference large enough that 2 people have only
remote chance of exactly same pattern
• Other repeated DNA sequences (VNTR) used:
people only two bands; probe for several loci for
statistical significance
5-16
DNA Fingerprinting
Like Southern blot
• Cut DNA with restriction enzyme
– Ideally cut either side of
minisatellite, not inside
• Run digest on gel, blot
• Probe with minisatellite DNA
Real samples
very complex
(Fig. 13)
5-17
Tandem repeat polymorphisms
Fig 2.25:
Different
numbers of
repeats can be
distinguished
by PCR using
primers to
flanking DNA
Or by Southern
with cutting
DNA
Forensic Uses of DNA
Fingerprinting
• People different DNA fingerprints:
pattern inherited Mendelian
fashion
– Establish parentage; identify
criminals; clear innocent people
• Actual pattern many bands;
smear together indistinguishably
– Forensics uses several probes for
single loci; places where many
alleles are possible (SSR, VNTR)
– Set of probes gives set of simple
patterns
Fig. 15
5-19
In Situ Hybridization: Locating Genes in
Chromosomes
• Labeled probes hybridize to
specific genes on chromosomes:
– Spread chromosomes from cell
arrested in metaphase
– Partially denature DNA, creates
single-stranded regions,
– hybridizes to labeled probe
– Stain chromosomes, detect
presence of label on chromosome
• Probe can be detected with
fluorescent antibody in technique
called fluorescence in situ
hybridization (FISH)
jjjjjjjjj
Fig. 16
5-20
Immunoblots (Western blots) – can quantify
Similar process to Southern blots, but proteins:
–
–
–
–
Electrophoresis of proteins
Blot proteins from gel to membrane
Detect protein using primary antibody to target protein
Labeled secondary antibody binds first antibody and
increases signal (can be nonradioactive)
Fig. 17
5-21
DNA Sequencing
• Modern DNA sequencing based on Sanger method:
• Dideoxy nucleotides terminate DNA synthesis
– 4 reactions, lots of 3 dNTPs,1 ddNTP in each:
• ddTTP reaction has some dTTP, lots of dATP, dCTP, dGTP
– Series of DNA fragments whose size is accurately
measured by electrophoresis (high % PAG)
– Last base in each fragment is known, that dideoxy
nucleotide used to terminate the reaction
– Ordering fragments by size tells base sequence of DNA
that was synthesized
5-22
Sanger
dideoxy DNA
Sequencing
(is DNA
synthesis)
High resolution
PAG gels
distinguish
fragments that
differ in size by 1
base;
Fig. 18
5-23
Automated dideoxy
DNA Sequencing
Dideoxynucleotides tagged with
different fluorescent molecules:
• Products of each
dideoxynucleotide termination
fluoresce different color
• Four reactions completed,
mixed, run on one lane of gel
• Detector reads colors and
calculates sequence
Fig. 20
5-24
5.4 Mapping and Quantifying Transcripts
• Mapping (locating start and end);
• Quantifying (how much transcript at a set time)
• Transcripts often not uniform terminator -: continuum
of species smeared on gel
• Techniques specific for sequence of interest
• Nuclease S1 mapping locates 5’ and 3’ ends (later)
5-25
Northern Blots
quantify RNA
With cloned cDNA, ask:
– How is gene expressed in different tissues?
• Run RNA from tissues on agarose gel, blot to membrane
• Hybridize to labeled cDNA or other probe
– Northern tells abundance of transcript
– Northern tells size of transcript
– Quantify using densitometer (Fig. 26)
5-26
5.5 Reporter Gene Transcription
• Place (surrogate) reporter gene under control of
specific promoter, measure accumulation of product
of reporter gene -> protein = gene expression
• Reporter genes chosen to have products convenient
to assay
– lacZ produces b-galactosidase, makes blue
cleavage product with Xgal (BCIG) substrate
– cat produces chloramphenicol acetyl transferase
(CAT) which inactivates Chloramphenicol
– Luciferase (luc) produces chemiluminescent
compound that emits light (detect luminometer)
5-27
Measuring Protein Accumulation in Vivo
• Gene activity in different tissues, different times,
monitored by measuring accumulation of protein (the
ultimate gene product)
• Two methods measure protein accumulation
– Immunoblotting / Western blotting uses
antibodies to detect proteins separated on gels
– Immunoprecipitation uses antibodies to
precipitate protein of interest from solution, test
what other proteins, DNA or RNA co-precipitate.
5-28
Review questions
1. Illustrate principle of DNA gel electrophoresis; indicate
comparative mobilities of DNAs with 150, 600, 1200 bp.
2. Compare process of Southern blot and RNA blot in terms
of process, and what information can be provided.
12. Diagram imaginary Sanger sequencing gel, and provide
DNA sequence.
21. Describe use of reporter gene to measure strength of a
promoter.
5-29