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Cloning and genetic
engineering
by
Ivo Frébort
Cloning
Clone: a collection of molecules or cells, all
identical to an original molecule or cell
To "clone a gene" is to make many copies of
it - for example, in a population of bacteria
 Gene can be an exact copy of a natural gene
 Gene can be an altered version of a natural
gene
 Recombinant DNA technology makes it
possible
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Plasmids
Naturally occurring extrachromosomal DNA
Plasmids are circular dsDNA
 Plasmids can be cleaved by restriction
enzymes, leaving sticky ends
 Artificial plasmids can be constructed by
linking new DNA fragments to the sticky
ends of plasmid
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Cloning Vectors
Plasmids that can be modified to carry
new genes
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Plasmids useful as cloning vectors must have
– a replicator (origin of replication)
– a selectable marker (antibiotic resistance
gene)
– a cloning site (site where insertion of
foreign DNA will not disrupt replication or
inactivate essential markers
DNA Libraries
Sets of cloned DNA fragments that together
represent the genes of a particular organism
Any particular gene may represent a tiny, tiny
fraction of the DNA in a given cell
 Can't isolate it directly
 Trick is to find the fragment or fragments in
the library that contain the desired gene
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Colony Hybridization
Disk is treated with base or heated to
convert dsDNA to ssDNA and incubated
with probes
 Colonies that bind probe hold the
fragment of interest
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Colony Hybridization
Southern Blots
The Polymerase Chain Reaction
What if you don't have enough DNA for colony
hybridization or Southern blots?
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The small sample of DNA serves as template
for DNA polymerase
Make complementary primers
Add primers in more than 1000-fold excess
Heat to make ssDNA, then cool
Run DNA polymerase (usually Taq)
Repeat heating, cooling, polymerase cycle
DNA sequencing
Chemical Cleavage Method
Four reactions are used
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G-specific cleavage with dimethyl sulfate,
followed by strand scission with piperidine
G/A cleavage: depurination with mild acid,
followed by piperidine
C/T cleavage: ring hydrolysis by hydrazine,
followed by piperidine
C cleavage: same method (hydrazine and
piperidine), but high salt protects T residues
DNA sequencing
Chain Termination Method
Based on DNA polymerase reaction
Run four separate reactions
 Each reaction mixture contains dATP,
dGTP, dCTP and dTTP, one of which is
P-32-labelled
 Each reaction also contains a small
amount of one dideoxynucleotide:
either ddATP, ddGTP, ddCTP or ddTTP
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Recombinant Proteins
Plant transformation
Common Strategies for Introducting Foreign Genes into Plant Cells:
 Agrobacterium infection
 Particle bombardment
 Electroporation
http://www.hort.purdue.edu/hort/courses/HORT250/animations/Leaf%20Disk%20Animation/leafdisk1.html
Regeneration of transgenic plant
from crown-gall callus
Tobacco plants overexpressing HvCKX genes
Transgenic Animals
Genes can be introduced into animals by
transfection - injection of plasmid DNA
into recipient cells
 Plasmids can be injected into fertilized
eggs in mice
 Expression is usually variable, because the
gene is inserted randomly
 Growth hormone transfection produces
mice that are very large!
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