PREPARED BY :MARWIN KUMAR

:NEOH KAI CHYUEN

:ELEANORA WONG
:SYAFIQULLAH KHAN
:TAN JING EE
:SITI NUR ZULAIQHA
LECTURER
:MADAM NOR HAYATI
SUBJECT
:LIFE SCIENCES

RDNA/ RDNA tech

Restriction enzymes/endonucleases
Plasmid
DNA ligase
Vectors
Bacteriophages
DNA library

DNA that has been created artificially (not

natural). DNA from two or more sources is
incorporated into a single recombinant molecule

Procedures by which DNA from different

species can be isolated, cut and spliced
together
New "recombinant " molecules are then
multiplied in quantity in populations of rapidly
dividing cells (e.g. bacteria, yeast).
Uses methods derived from biochemistry of
nucleic acids coupled with genetic techniques
originally used for the study of bacteria and
viruses

1. A gene is located on a chromosome map

2. A DNA library is constructed plasmid are
obtained, cleavage occurs and RDNA prod.
3. The gene of interest is isolated (cloned) from
the library via plasmid DNA isolation, restriction
digestion and electrophoresis.
4. Multiple copies of gene interest are produced
for study.

Restriction enzymes cuts DNA

at specific sites (restriction fragments)

DNA ligase pastes the DNA

fragments together

The cut segments are inserted into other DNA

molecules that serves as vectors(PLASMID)

DNA cutting enzymes. Restriction

endonucleases
cuts DNA at a specific site defined by a
sequence of bases in the DNA (recog.site)
forming sticky ends (ss)
Palindromic sites
Eg. BamHI cuts 5' GGATCC 3'
3' CCTAGG5 Eg.2 HaeIII cuts 5'GGCC3'
3'CCGG5'
several hundred endonucleases have been
extracted from bacteria and many are used in
recombinant DNA research. eg EcoR1,Hind III,
HaeIII, TaqA1, Sau3A

BACTERIAL ORIGIN=ENZYME WHICH CLEAVE FOREIGN DNA AS

IT(ECORI;ESCHERICHIA COLI) PROTECT BACTERIA FROM
BACTERIOPHAGE INFECTION

Carrier DNA mol = transfers the RDNA into the

host cell.
Within host cells, vectors can replicate producing
many DNA segments = identical copies
(CLONES)
Host cells pass the recombinant DNA mol on their
progeny = population of cells.
Cloned DNA segments recovered from host cells
for purification and analysis

Molecules of DNA that are found in bacteria

Act as a system to transfer genetic material to
other bacteria, allowing those to express the
transmitted genes.
small (a few thousand base pairs)
usually carry only one or a few genes
circular
have a single origin of replication

Typical w&b screening procedure

Schematic
represent typical
plasmic vector
which can be use
for w&b screening
test.

Because each cloned DNA segement is relatively

small, many separate clones must be constructed .
A set of cloned DNA segments derived from a
single individual represents a library
Cloned libraries could be an entire genome, a
single chromosome, or a set og genes compiled
together in a single cell type.

Applications of recombinant DNA

technology
Used widely in research and hospital laboratories.
Broad applications - medicine, agriculture,

Medicine production of industrial and commercial compounds

Insulin Diabetes
drugs (angiostation and endostatin) ,
Factor VIII Haemophilia A
Factor IX Haemophilia B
EPO Anaemia
Interleukins and interferons
Tissue plasminogen activator dissolve blood clots
Hormones = GH, parathyroid
Oxytocin
Adenosine deaminase sev. Com. Imm (SCID)

Diagnostic kits Hep, AIDS

Antibiotics

genetic testing,Mapping the chromosomal

location of genetic disorders. RFLP, DNA
fingerprinting the HG project
Gene therapy manipulation of DNA to tx
diseases by altering individuals genes. (CF,
PKU, DMD) ETHICAL ISSUES
Forensic applications - All individuals are
genetically unique = a distinct "genetic
fingerprint, all types of specs, old and new
Animals = models of genetic diseases Eg GE mice

Vaccines :vare
:v created by transferring the
genes that determine a pathogen's surface
configuration to a microorganism.
When the GM microorganism is used in a
vaccine, its surface stimulates the production of
antibodies.
These antibodies protects an individual against
the pathogen.
Successful vaccines have been produced for
influenza, cold sores, and hepatitis B(HBsAg).

Animals improved quality/quantity

of meat products
Plants Gene altered plants :
improved crops quantity, quality
and pest resistance. Eg golden rice
GM to contain beta carotene vit A
deficiency.