Cloning and genetic

engineering

by
Ivo Frbort

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
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
Cloning Vectors
Plasmids that can be modified to carry
new genes
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
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
Colony Hybridization
Southern Blots
The Polymerase Chain Reaction
What if you don't have enough DNA for colony
hybridization or Southern blots?
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
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
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!