Molecular Biology Course Outline
2007
Book: Molecular Biology, 4th edition, McGraw Hill
by Robert Weaver
Molecular and Cell Biology I (Wednesdays 3:00 -6:00 pm)
由謝明麗,劉薏雯,蔡世峰教授合授
Week Date
Topic
Instructor
1 9/19
2 9/26
3 10/3
4 10/10
Molecular tools for studying Gene activity
Molecular tools for studying Gene activity (II)
The transcription apparatus of Prokaryotes
Operons: Major shift in Prokaryotic transcription
謝明麗
謝明麗
謝明麗
謝明麗
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•
•
•
•
•
•
•
•
•
•
•
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5
6
7
8
9
10
11
12
13
14
15
16
17
10/17
10/24
10/31
11/7
11/14
11/21
11/28
12/5
12/12
12/19
12/26
1/2
1/9
Eukaryotic RNA polymerases and their promoters
General transcription factors in Eukaryotes
Transcription activators in Eukaryotes
Message RNA processing: Splicing
Message RNA processing: Capping and polyadenylation
Mid-term
The Human Genome Project and the HapMap Project
Genomic technology, microarray, and proteomics
Cancer genomics, microbial genomics, and pharmacogenomics
Model organisms and systems biology
特別演講
特別演講
Overview
劉薏雯
劉薏雯
劉薏雯
劉薏雯
劉薏雯
劉薏雯
蔡世峰
蔡世峰
蔡世峰
喻秋華
Molecular Tools for Studying Genes and
Gene Activity
Molecular Separation
Gel Electrophoresis
• Gel electrophoresis is used to separate
different species of:
– Nucleic acid
– Protein
5-5
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
5-6
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
5-7
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
5-8
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)
5-9
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
5-10
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
5-12
Two-Dimensional Gel Electrophoresis
Ion-Exchange Chromatography
• Uses a resin to separate substrances according to
their charges
• DEAE-Sephadex chromotography uses an ionexchange resin that contains positively charged
diethylaminorthyl (DEAE) group.
• These positive charges attract negatively charged
substances, including proteins.
• Phosphocellular is commonly used negatively
charged resin.
Gel Filtration Chromatography
• Uses columns filled with porous resins that
let in smaller substances, but exclude larger
ones.
• The smaller substances are slowed in their
journey through the column, but larger
substances travel relatively rapidly through
the column.
Tracers
Detection
Labeled tracers
(e.g. 3H, 14C, 32P, 35S, 125I)
Autoradiography using x-ray film
phosphorimaging,
liquid scintillation counting
Non-radioactive tracers
(e.g. fluorochrome, hapten)
Fluorescence microscope
Enzyme-couple chemiluminescence
Autoradiography or
phosphorimaging
Enzyme-couple chromogenic
Nucleic Acid hybridization
Southern blot
DNA : DNA
DNA fingerprinting and DNA typing
DNA : DNA
Colony hybridization
DNA : DNA
Northern blot
RNA : cDNA
In situ hybridization
(e.g fluorescence in situ hybridization; FISH)
Chromosomal DNA :
DNA
Microarray
DNA: DNA
cDNA : cDNA
Southern blots
RFLP (Restriction Fragment Length Polymorphisms)
DNA Testing by Allele-Specific Cleavage
DNA Testing by Allele-Specific Oligonucleotide hybridization
DNA fingerprinting
DNA typing
Northern blots (measuring gene activity)
FISH
(Fluorescence
in situ Hybridization)
Locating genes in
chromosomes
22q11.12
Gene chips
(Microarray)
Gene identification
Southern blot
FISH
Immunoblots (Western Blots)
DNA sequencing
Restriction mapping
Restriction mapping
(physical mapping)
Identification of a new gene
Identification of the transcript
mapping the start site and stop site
measuring active transcripts
Identification of the gene product
quantitative
and
qualitative
Immunoblots
(Western
blots) analysis
Identification of the gene function
gain of function
loss of function
Mapping the start site of transcripts
S1 mapping
Primer extension
Run-off transcription
S1 mapping the 5’ end
Primer extension
Run-off transcription
Mapping the stop site of transcripts
S1 mapping
S1 mapping the 3’ end
Measuring active transcripts
Northern blot
In situ Histochemistry stain
Nuclear run-on transcription
Nuclear Run-on transcription
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
5-46
Western Blots
5-47
Qualitative analysis of
the cis/trans element activity
Reporter gene activity
Cellulose filter binding assay
Gel mobility shift assay
DNase footprinting
DMS footprinting
Reporter gene
Gel mobility shift assay
DNase footprinting
DMS footprinting
Gain of function
Transgenic clone
Reporter gene activity
Transgenic clones
Loss of function
Site-directed mutagenesis
Knockout mouse
Site-directed mutagenesis
By oligonucleotide
Making a knockout
mouse
Finding RNA sequences that
interact with other molecules
• SELEX (systematic evolution of ligands by
exponential enrichment)
• RNAs that interact with a target molecule
are selected by affinity chromatography,
then converted to double-stranded DNAs
and amplified by PCR.
Fig. 5.39