Molecular Biology Course Outline 2007

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