Medical Biophysics graduate course

1
Medical Biophysics graduate course MBP
518A Molecular Imaging Lecture 5 Instructor
Dr. Donna Goldhawk Basic Techniques in
Molecular Biology
2
Contact Information Dr. Donna Goldhawk Imaging
Program Lawson Health Research Institute Office
E4-151 Labs F4-127a / F5-119 519-685-8500 ext.
64863 Donna.Goldhawk_at_lawsonimaging.ca
3
Imaging
Molecular Imaging
Molecular Biology
4
Cloning DNA Insert-vector Hybrid Restriction
Enzymes Transformation Selection Analysis Po
lymerase Chain Reaction Sequencing Site-direct
ed Mutagenesis Gene Expression Transfection V
irus-mediated Gene Delivery Analysis Antibody
Structure Promoters Reporter Genes
5
Reference Molecular Cloning a laboratory
manual Sambrook, J. and Russell, D.W. 2001 3
volumes Cold Spring Harbor Laboratory
Press Cold Spring Harbor, NY
6
What are the main steps involved in cloning?
DNA sequence Cloning vector Host cell
7
Vector
DNA insert
Ligation
DNA construct
Transformation Selection
Host cell
Culture
8
DNA sequence entire gene or fragment restrictio
n digestion to linearize to provide
suitable ends for insertion Cloning
vector bacterial plasmid extrachromosomal DNA
that replicates autonomously closed, circular
DNA restriction digestion to
linearize to provide suitable ends for
insertion Host cell Bacterium prokaryote E
scherichia coli E. coli growth subject to
antibiotic resistance Yeast eukaryote Sacc
hromyces cerevisiaie budding yeast subject
to auxotrophic growth
9
Cloning Enzymes Restriction endonucleases Eco
RI recognition site G AATTC Multicloning Site
(MCS) Alkaline phosphatase Removes the 5
phosphate Prevent vector recircularization Pro
mote ligation between insert and vector T4 DNA
ligase Catalyzes the formation of
phosphodiester bonds 5 phosphate 3
hydroxyl Cohesive ends align the DNA Blunt
end ligation Replication seals the nicked DNA
10
Restriction Enzymes Bacterial enzymes Type II
restriction endonucleases Cleave palindromic
sequences Ex. Eco RI Nomenclature genus Esc
herichia E species coli co strain (opti
onal) R order of characterization I Ex. Hpa
I Hpa II - Haemophilus parainfluenzae -
first and second type II restriction enzymes
isolated
11
Cleavage sites of some restriction enzymes Eco
RI G AATTC Bam HI G GATCC Sal I G TCGAC Xho
I C TCGAG Hpa I GTT AAC Hpa II C CGG
12
(No Transcript)
13
Cloning Vector
C
14
Cloning Enzymes Restriction endonucleases Eco
RI recognition site G AATTC Multicloning Site
(MCS) Alkaline phosphatase Removes the 5
phosphate Prevent vector recircularization Pro
mote ligation between insert and vector T4 DNA
ligase Catalyzes the formation of
phosphodiester bonds 5 phosphate 3
hydroxyl Cohesive ends align the DNA Blunt
end ligation Replication seals the nicked DNA
15
Factors to consider Reannealing of the cut
vector Treat with alkaline phosphatase (AP) -
calf intestinal AP (CAP or CIP) - to prevent
recircularization of the vector - to promote
ligation between insert and vector Ligation
reaction T4 DNA ligase 15oC overnight
incubation Why are these reaction conditions
advantageous for creating a hybrid vector-DNA
construct?
16
What are the main steps involved in cloning?
DNA sequence Cloning vector Host cell
17
Host cell Bacterium prokaryote Escherichia
coli E. coli growth subject to antibiotic
resistance Yeast eukaryote Sacchromyces
cerevisiaie budding yeast subject to
auxotrophic growth Growth environment agar
plate liquid culture Carbon source ex.
tryptone Amino acids ex. yeast
extract NaCl

18

• Host cell characteristics
• Recombination negative RecA-
• -lack restriction endonucleases
• -competent
• capable of taking up DNA
• at high cell density
• under starvation conditions
• not usually intrinsic
• induced

19

• Transformation
• 1. Chemical method
• mid-log phase cells
• CaCl2 suspension, stored at -70oC
• chill on ice
• heat shock at 42oC
• creates transient holes in the cell wall
• 2. Electroporation
• mid-log phase cells
• washed in low salt buffer
• resuspended in 10 glycerol, high density,
  stored at -70oC
• chill on ice
• apparatus disperses a discrete charge to the
  DNA sample
• - set in a chamber outfitted with electrodes
• electric field-mediated membrane
  permeabilization

20
Transformed cells on an agar plate Selectio
n of colonies bearing the construct of
interest Antibiotic Resistance conferred by
vector Ampr Kanr
21
1. Ampr produces b-lactamase disables
ampicillin by cleaving its b-lactam ring -
ampicillin blocks synthesis of the peptidoglyc
an layer that lies between the inner and
outer cell membranes 2. Kanr produces
Kanamycin phosphotransferase disables kanamycin
by phosphorylation - kanamycin blocks protein
synthesis by binding irreversibly to the
30S ribosomal subunit
22
(No Transcript)
23
Transformation versus Transfection
Bacteria are transformed. Mammalian cells are
transfected with plasmid vectors or
transduced with viral vectors. TRANSFECTION S
everal methods Lipofectamine 2000 TRANSDUCTION
DNA is packaged in a virus particle.
24
Virus-mediated gene delivery Mechanism of
action 1. bind to a specific cell
type 2. internalize the vector
DNA 3. transport the DNA to the
nucleus 4. replicate the vector
DNA 5. encapsidate the DNA 6. release the
viral particles 7. repeat the life cycle
25
HIV Life Cycle
26
(No Transcript)
27
Antigen (Ag) a foreign substance that
stimulates an immune response Antibody (Ab) a
protective protein that recognizes and
helps body cells destroy foreign
material Monoclonal Antibody (MAb) recognizes
one antigenic site consists of one type of
Ab therapeutic agent
28
(No Transcript)
29
(No Transcript)