Download Restriction Enzyme

Chapter 5
Exploring Genes and Genomes
Basic Essential Tools for Gene Exploration
1. Restriction Enzymes : Precise Sequence-Specific Cleavage of DNA
2. Blotting Techniques : Southern and Northern Blotting
3. DNA Sequencing : Determination Precise Nucleotide Sequence of DNA
(human 3 billiion base-pairs)
4. Solid-Phase Synthesis of Nucleic Acids : Oligo(deoxy)nucleotide
5. Polymerase Chain Reaction (PCR) : Amplification of a segment of DNA
(Nobel Prize in Chemistry 1993 Dr Kary B. Mullis, U.S.A., for his
invention of the polymerase chain reaction (PCR) method)
6. Computerized Analysis : Bioinformatics
Restriction Endonuclease (Restriction Enzyme)
• Found in a variety of prokaryotes.
• Foreign DNA cleavage.
• Self DNA protection by methylation
Palindromic
Inverted repeat,
Usually
4-8 base pairs
• Sequence-specific cleavage
• Hydrolysis of a phosphodiester bond
• Palindromic sequence recognition
• Staggered or even cutting
• 1978 Hamilton Smith, Daniel Nathans
Recognition
Site for Sac I
(Streptomyces
achromogenes)
Rule of Naming Restriction Enzymes
• First three letters for the host organism (e.g. Hin for Haemophilus influenza)
• Next one letter for strain designation, if necessary (e.g. HinD for Haemophilus influenza D)
• Last one letter for a number, if more than one restriction enzyme have been identified from
the same species. (e.g. HinD III for the third RE found in Haemophilus influenza D)
Visualization of DNA Fragments by Gel Electrophoresis
Non-Denaturing Gel Electrophoresis Systems
For < 1 k bps : Polyacrylamide Gel Electrophoresis
For < 20 k bps : Agarose Gel Electrophoresis
Pulsed-Field Gel Electrophoresis (PFEG) can separate
DNA molecules in the range of million base pairs.
Denaturing Gel Electrophoresis Systems
Urea Polyacrylamide Gel can distinguish DNA fragments
differing in length just by one nucleotide out of several
hundred base pairs
EtBr (ethidium bromide) Staining of Nucleic Acids;
intercalation into base stacks; fluorescence upon UV light;
50 ng of DNA is detection limit
Ethidium bromide
intercalating agent commonly used for nucleic acid staining
may be a mutagen, carcinogen
Southern Blotting for DNA & Northern Blotting for RNA
Denaturation
By formaldehyde
Hybridization Probes
Hybridization
b-rays from
radioisotope
for Southern Blots : single-stranded DNA
for Northern Blots : single-stranded mRNA
Sanger Di-Deoxy Method for DNA Sequencing
• Partial termination of DNA polymerase reaction
by including dideoxy nucleotides
• Urea Polyacrylamide Gel Electrophoresis
• Visualized by autoradiography or fluorescence
Automated Solid-Phase DNA Synthesis
Deoxyribonucleoside 3’-phosphoramidite : Incoming Nucleotide
DMT : Dimethoxytrityl Group ; Protect 5’ hydroxyl group of incoming nucleotide
bCE : b-Cyanoethyl Group ; Protect 3’ phosphoryl oxygen
All bases are protected during synthesis.
Phosphotriester is oxidized by iodide to make phosphodiester.
Dichloroacetic acid removes DMT to generate a free 5’ hydroxyl group.
Each cycle can be completed within 10 min and the efficiency is better than 98%.
At the end of the synthesis, NH3 is added to remove all the protecting groups.
By HPLC, choose longest one.
Polymerase Chain Reaction (PCR) :
Amplification of Specific DNA Sequences
Required Components
• Pair of primers that hybridize the target
• All four deoxyribonucleotides (dNTPs)
• A heat stable DNA polymerase
• Target sequence (template)
Cycling Reactions
• Strand Separation (Denaturation): 95
• Hybridization of Primers (Annealing): 54
• DNA Synthesis (Extension): 72
• Repeats the above three steps
Parent DNA annealing?
Theoretical PCR
Amplification Fold = 2n
(n : cycle number)
Thus,
Million Fold Amplification
after 20 Cycles, and
Billion Fold Amplification
after 30 Cycles
PCR machine
PCR Application
1. Medical Diagnostics
- Detection of pathogen (bacteria
and virus)
- Detection of cancers (mutations of
ras genes)
2. Forensics
- Some genes are highly variable
within a population (human leukocyte
antigen type, HLA)
3. Molecular Evolution
- DNA is very stable and remain intact
for thousands of years or longer,
particularly when shield from air, light
and water
Vector
• Autonomously replicable DNA in an
appropriate host.
• Delivering vehicle for the gene of
interest into a host
Insert
• DNA fragment of the gene of interest
Restriction Enzyme
• To generate joinable DNA fragments
Ligase
• To join the cut DNA fragments
Linker
• Small DNA fragment containing
restriction enzyme sites
• Can be attached to any DNA fragment
by a ligase and cut by a particular
restriction enzyme to generate
specifically desired cohesive ends
Plasmids
• Naturally occurring circular DNAs
acting as accessory chromosomes
(small extra chromosomes) found
in bacteria; episome, disposable
• Plasmids can be autonomously
replicated
in
independently
of
bacteria
the
host
chromosome.
• Plasmids
utilized
in
molecular
cloning in the laboratory usually
contain cloning sites and selection
markers (e.g. antibiotics resistance
genes).
Useful as a cloning site
Plasmids
Evolved…
• Polylinker Region
 More Cloning Sites
• Further
Selection
Recombinants
 X-gal Color Selection
• Stronger Replication Origin
 More DNA Synthesis
for
Bacteriophage l
• Bacteriophage l  Inserts up to 10 kb
arms
• Cosmid : A Circular Vector with Bacteriophage
Backbone  Inserts up to 45 kb (hybrid of
plasmid/ l phase)
• BAC : Bacterial Artificial Chromosome
 inserts up to 300 kb
• YAC : Yeast Artificial Chromosome
 inserts up to 1000 kb
YAC
Chromosome Walking
Efficient Analysis of Long Stretches of DNA
Construction of
l-Phage Genomic Library
Cloning of a Gene
from
Genomic Library
Genomic Library Screening
with a Radioactive Probe Containing
the Sequence of the Gene of Interest
32P-labeled
probe
How to Make a Specific Probe for Library Screening ?
Deduce the Possible Nucleotide Sequences from Amino Acid Sequence
 Synthesize Oligonucleotides Containing Corresponding Sequences
Make cDNA from mRNA by using Reverse Transcriptase
 PCR using cDNA Templates and Specific Primers for the Gene of Interest
cDNA Library Construction
Poly(A) mRNA Isolation
using Oligo(dT) Column
Synthesis of Double-Stranded cDNA
(Total cDNA)
Linker Ligation
Cloning of Total cDNA
into Plasmid (or Bacteriophage Vectors)
Transform (or Infect) Host Bacteria
Obtain Pool of Bacteria (or Bacteriophage)
cDNA Library
cDNA Library Screening
Mutation of DNA  Generation of New Proteins
• Deletions : Single Cleavage by RE
 Exonuclease Digestion  Ligation
• Substitutions : Oligonucleotide-Directed Mutagenesis
Digestion of template strand by Dpn1 enzyme
• Insertions : Cassette Mutagenesis
• Designer Genes : Chimeric Proteins
(cf. Immuno-Toxin Therapy for Cancer)
Classical Random Mutant Screening
(Forward Genetics)
vs.
Generation of a Specific Mutant by Design
(Reverse Genetics)
Complete
Genome
Sequencing
Gene # ?
Efficiency ?
Comparative
Genomics
Evolution ?
Comprehensive Gene Expression Analysis Using Microarray
• Labeling Total cDNAs from Sample 1
with Green Fluorescence
• Labeling Total cDNAs from Sample 2
with Red Fluorescence
• Mix Two Pools of cDNAs
• Hybridize against the Gene Chip
• Variations in Gene Expression Profile
Can Be Visualized.
Breast Cancer Specific
Changes in Gene Expression
Figure out!
Animation!
http://www.bio.davidson.edu/Courses/genomics/chip/chip.html
Expression of Foreign Genes in Host Cells
•+oligo T primer
•High pH
•Terminal
transferase,
add dG to 3’
+synthetic
linker
Expression of a Gene of Interest from Genomic DNA ???
Introns…
Splicing Apparatus Is Absent in Prokaryotes…
Introns Are Already Removed in cDNAs Made from Mature mRNAs
Expression of Foreign Genes in Mammalian Cells
• Necessity : Post-translational Modification
Deficiency in Bacteria
• Transfection
Calcium Phosphate Precipitation
Liposome-Mediated Vesicle Fusion
(cf. Stable vs. Transient Transfection)
• Micro-Injection into the Nucleus
• Viral Infection
DNA Virus : Adenovirus, etc. (Transient Expression)
Retro-Virus : Moloney Murine Leukemia Virus (MMLV),
Lenti-Virus, etc. (Stable Expression)
Vaccinia Virus : Viral DNA Replication in the Cytoplasm
Shut Down Host Protein Synthesis
• Baculovirus : Infection into Insect Cells (Sf9),
Efficient Protein Production Factory
Transgenic Animals Can Express Exogenous Genes of Interest
Growth Hormone Deficiency  Dwarfism
Growth Hormone Excess  Gigantism
• Metallothionein: heavy metal binding
protein with many cysteins
• Growth hormone induction (500x) by
adding cadmium into drinking water
• Microinjection of several hundred copies
of GH expressing plasmid into the male
pronucleus of a fertilized mouse egg 
insertion into the foster female uterus
• Check by southern blotting
Gene Disruption in Animal (Knock-Out Animal)
(or Markers)
Myogenin(+/+) Mouse Muscle
Targeted Gene Disruption
by Homologous Recombination
Myogenin(-/-) Mouse Muscle
Gene Knock-Out (or Knock-In) Technology Has Enabled Us to Address
In Vivo Functions of a Gene of Interest at the Level of a Whole Organism.
Gene Expression Knockdown
by RNA Interference
• Double stranded RNA cleavage by Dicer
• 21 nucleotide double stranded RNA
(siRNA : short interfering RNA)
• Recognition by RISC
(RNA Induced Silencing Complex)
• Hybridization with mRNA
• mRNA cleavage
• C.elegans, E.coli
Ti (Tumor Inducing) Plasmids
Can Deliver Foreign Genes into Plants
Dicot, monocot
Crown Gal
Plant Tumor Caused by
Agrobacterium tumafaciens
A small synthetic plasmid carrying the gene of
interest in T-DNA fragment can be added to
Agrobacterium colonies harboring naturally
occurring Ti plasmids, then, by recombination,
recombinant Ti plasmid can be generated.
More Methods for
Gene Transfer into Plants
Electroporation
• Make protoplast by using cellulase
• High electric pulse makes
membranes transiently permeable.
• Plasmid DNA can enter the cells.
• Marker selection
Gene Guns
• DNA is coated onto 1-mm-diameter
tungsten pellets
• These microinjectiles are fired at
the target cells with a velocity
greater than 400 m s-1.
GMOs in wheat, soybean, corn, and rice; most efficient way
Mutual Re-Enforcement of Protein and Nucleic Acid Chemistry