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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 - PowerPoint PPT Presentation
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Molecular Biology Course Outline 2007
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 Molecular tools for studying Gene activity 謝明麗2 9/26 Molecular tools for studying Gene activity (II) 謝明麗3 10/3 The transcription apparatus of Prokaryotes 謝明麗4 10/10 Operons: Major shift in Prokaryotic transcription 謝明麗
Book: Molecular Biology, 4th edition, McGraw Hill by Robert Weaver
• 5 10/17 Eukaryotic RNA polymerases and their promoters 劉薏雯• 6 10/24 General transcription factors in Eukaryotes 劉薏雯• 7 10/31 Transcription activators in Eukaryotes 劉薏雯• 8 11/7 Message RNA processing: Splicing 劉薏雯• 9 11/14 Message RNA processing: Capping and polyadenylation 劉薏雯• 10 11/21 Mid-term 劉薏雯• 11 11/28 The Human Genome Project and the HapMap Project 蔡世峰
• 12 12/5 Genomic technology, microarray, and proteomics 蔡世峰• 13 12/12 Cancer genomics, microbial genomics, and pharmacogenomics 蔡世峰• 14 12/19 Model organisms and systems biology
喻秋華 • 15 12/26 特別演講• 16 1/2 特別演講• 17 1/9 Overview
Molecular Tools for Studying Genes and Molecular Tools for Studying Genes and Gene ActivityGene Activity
Molecular Separation
5-5
Gel Electrophoresis
• Gel electrophoresis is used to separate different species of: – Nucleic acid– Protein
5-6
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
5-7
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
5-8
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
5-9
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)
5-10
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
5-12
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
Two-Dimensional Gel Electrophoresis
Ion-Exchange Chromatography
• Uses a resin to separate substrances according to their charges
• DEAE-Sephadex chromotography uses an ion-exchange 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.
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
Tracers Detection
In situ hybridization(e.g fluorescence in situ hybridization; FISH)
Nucleic Acid hybridization
DNA : DNA
Southern blot DNA : DNA
DNA fingerprinting and DNA typing
Colony hybridization DNA : DNA
Northern blot RNA : cDNA
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 transcriptmapping the start site and stop sitemeasuring active transcripts
Identification of the gene productquantitative and qualitative analysis
Identification of the gene functiongain of functionloss of function
Immunoblots (Western blots)
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
5-46
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
5-47
Western Blots
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