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Chapter 5
Part III. Recombinant DNA technology
• Cloning strategies
• Polymerase chain reaction (PCR)
• Applications
Recombinant DNA technology
(Gene cloning, molecular cloning, genetic engineering)
Methodology for transferring genetic information
(genes) from one organism to another
• Characterization of the genes
• Large production of proteins
• Mutants
Tools of recombinant DNA technology
• Restriction endonucleases - cut DNA at
specific sites
• DNA ligase or other DNA modifying enzymes
• Cloning vectors - DNA molecules that can be
replicated
• Reporter genes
• Model organisms
Restriction enzymes
•
•
Bacteria’s “immune system” for
protection from infection by foreign DNA
Three types
– Type I and Type III : Both the endonuclease
and the methylase activity; Remote
recognition site
– Type II : Only endonuclease activity;
Specific and predictable recognition;
•
Cohesive (sticky) or blunt ends
Palindromic restriction sites
Restriction map
Restriction map
Restriction-fragment length polymorphism
(RFLP)
Inheritance of RFLPs
Cloning vectors
• Plasmids
– Replication origin, selectable marker & polylinker
– Stringent control (low copy number) or relaxed
control (medium to high copy number)
• Viral vectors
– Bacteriophage λ, cosmid & M13 : Bacteria
– Baculoviruses: Insects
– Retroviruses, lentiviruses & adenoviruses:
Mammalian cells
• Yeast artificial chromosome (YAC) and bacterial
artificial chromosome (BAC)
Plasmid cloning vector
lacZα
Polylinker
(Multiple
Cloning
Site)
ampR
Replication
origin
Construction of recombinant DNA
Cloning in λ phages
Cloning strategies
•
•
•
•
•
•
DNA ligase
PCR
Terminal transferase
Adaptor
Topoisomerase
Recombinase
Cloning using DNA ligase
Cloning using terminal transferase
Cloning using synthetic adaptor
Reporter genes
• Selectable markers
– Antibiotic resistance
– Nutritional markers
• LacZ (β-galactosidase)
• Luciferase
• Green fluorescence protein (GFP)
Insertional gene inactivation
(Replica plating)
β-Galactosidase as a reporter
GFP as a reporter
GFP
Tsien at UCSD
Southern blot
Detection of specific DNA sequence
Colony (in situ) hybridization
Identification of the clones containing a DNA of interest
Model organisms
Polymerase Chain Reaction (PCR)
Technique for the exponential amplification
of a specific DNA segment
• Template DNA
• Two oligonucleotide primers
• Heat stable DNA polymerase
(Taq, Pfu etc)
Three step process
• Template denaturation
• Primer annealing (hybridization)
• Primer extension (polymerization)
Denaturing
Annealing
Extension
Heat stable DNA polymerase
dNTPs
Exponential amplification
Cycle
number
After 4 cycles
After 32 cycles
Number of the
target DNA
molecules
1
0
2
0
3
2
4
4
5
16
10
256
15
8,192
20
262,144
25
8,388,608
30
268,435,456
32
1,073,741,824
Application of PCR
• Clinical applications - Diagnosis of
infectious diseases and rare mutations
• Forensics – DNA fingerprinting
• Molecular archeology – Evolutionary study
• Asymmetric PCR – DNA sequencing
• Site-directed mutagenesis
Site-directed mutagenesis
Applications of
the recombinant DNA technology
• Recombinant proteins
– Research
– Medical purposes
• Genetically altered organisms
– Transgenic
– Knockout
• Gene therapy
Recombinant proteins
Genetically modified organisms (GMOs)
• Bacteria – Bioremidation, biomining,
biofuel etc
• Plants – Resistace to pests, herbicides or
harsh environmental conditions; improved
shelflife; increased nutritional value
• Animals – Transgenic or cloned animals
Insect-resistant cotton
GloFish
Golden rice
Giant mouse
Dolly, the cloned sheep
Approaches for gene therapy
• ex vivo – Treatment of cells with a vector
outside of the body
• in situ – Direct application of the vector to
affected tissues
• in vivo – Direct injection of the vector into
the blood stream