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TECHNOLOGY
GENETIC ENGINEERING
TECHNOLOGY THAT MANIPULATES OR
MODIFIES THE GENE OF AN ORGANISM,
WITH THE INTENTION OF CREATING
IMPROVED PRODUCTS OR METHODS.
TRANSGENIC PLANT/ANIMAL
RE
VECTORS
DNA LIGASE
HOST O/M
RESTRICTION
ENDONUCLEASES/ ENZYMES
Recombinant DNA technology developed from the
discovery of certain bacterial strains which seemed
immune to attack by bacteriophages.
These strains produced special enzymes that
restricted the replication of foreign DNA which had
infected the bacterial cells. (hence they were called
restriction enzyme)
The site at which a restriction enzyme acts is called
the restriction site. This site is typically 4-6
nucleotides long.
Blunt ended
DNA
Produced
staggered
cuts,
sticky
ends.
Restriction
site which
produces
sticky
ended DNA
after
cleavage palindromi
Sticky
ended DNA
EXAMPLES OF RESTRICTION
ENZYMES AND RESTRICTION SITES
VECTORS
DNA molecules, derived from a plasmid or
bacteriophage into which DNA fragments may be
inserted.
A. Definition - Bacteriophage (phage) are obligate
intracellular parasites that multiply inside bacteria
by making use of some or all of the host
biosynthetic machinery (i.e., viruses that infect
bacteria.).
A 'plasmid' is a DNA molecule separate from the
chromosomal DNA and capable of autonomous
replication. In many cases, It is typically circular and
double-stranded. It usually occurs in bacteria, and is
sometimes found in eukaryotic organisms (e.g., the
2-micrometre-ring in Saccharomyces cerevisiae).
Properties of Plasmids
1. Small molecules with known structures
2. Contain an origin of replication ---the
plasmid can replicate itself in the host cell
3. Have one or more restriction sites
recognised by RE
4. They code for special functions which
confer well-defined features on the host,
thus making them easier to be selected.
pBR322
pUC18
YAK
lambda phage
VECTORS
Cloning Vector
322..
Expression vectors
Expression Vectors
Ti vectors
- Naturally occuring plasmids (around 200 kb
in size
- -isolated from bacterium Agrobacterium
tumefaciens, which is a soil borne plant
pathogen that causes disease namely as
crown gall disease.
DNA LIGASE
Required to anneal or join the sugarphosphate
backbones
in
the
recombinant DNA molecule.
HOST O/M
CLONING
Cloning in recombinant technology is
defined as the production of a cell line or
culture, all of whose members contain
identical copies of particular nucleotide
sequence.
CLONING
Example :
The process of insulin production by
E. coli as an example.
Note that the chances of success
are very small !!!
STEP 1 (ISOLATION)
Two kinds of DNA are prepared;
1. the gene of interest (insulin gene) which
is cultured from human cells,
2. bacterial plasmid with two particular
genes, that is the ampR gene and the lac
Z gene.
. The ampR gene confers resistance to the
antibiotic ampicillin. Thus bacteria with
this plasmid can grow in media which
contain ampicillin.
STEP 2 (Recombination)
Half of the RE solution is mixed with the
human DNA
This will produce several hundred
thousand DNA fragments with sticky ends.
The rest of RE enzyme is then mixed with
the plasmid molecules and this will
produce an equally large number of linear
plasmids molecules with sticky ends
complementary to those of the target DNA
fragments.
STEP 3 (TRANSFORMATION)
The mixture of DNA molecules produced
in step 2 is added to a culture of bacterial
cells, usually E coli.
Calcium ions are added ---stimulated to
take up small pieces of DNA
Only about 1% -----recombinant DNA.
STEP 4 (CLONING)
The bacteria are plated onto nutrient
plates which contain ampicilin and
sugar called X-gal.
Untransformed strains of E. coli will be
killed by ampicilin. Only those cells
which have acquired the plasmid with
the ampR gene will be able to grow in
the medium.
P? Q? R?
STEP 5 (SCREENING)
A nitrocellulose membrane or filter paper is
laid on the surface of a nutrient agar plate
on which the bacteria colonies with
recombinant
plasdmids
have
been
cultured.
Bacteria cell will be transferred to the filter.
The filter is treated with sodium hydroxide
solution which denatures the DNA by
breaking the hydrogen bonds between the
two polynucleotide strands.
This leaves single-stranded DNA on the
filter. Single-stranded DNA will pair up
(hybridise) with any nucleic acid molecule
with a complementary sequene of bases.