Recombinant DNA Technology

Recombinant DNA technology
Essential vocabulary
Biotechnology/ GE
RDNA/ RDNA tech
Restriction enzymes/endonucleases
Cloning
DNA ligase
Vectors
Plasmid
Bacteriophages
DNA library
Biotechnology
The manipulation of biological processes and/or
organisms for the benefit of mankind
RDNA
DNA that has been created artificially (not
natural). DNA from two or more sources is
incorporated into a single recombinant
molecule
RDNA tech
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
RDNA tech process

1.
A gene is located on a chromosome map

(RFLP)
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.
steps
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
Vectors
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
Plasmids
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
Resctriction enzymes
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
Specific palindromic sites
Library construction
Because each cloned DNA segemnt 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
singe chromosome, or a set og genes compiled
together in a single cell type.
summary
Applications of recombinant DNA

technology
Used widely in research and hospital laboratories.
Broad applications - medicine, agriculture,
??..
1.
The processes of inheritance and gene

expression.
2. Process and tx of various diseases
3. Generation of economic benefits eg.
improved agricultural products.
Applications
Molecular
Biology/Research
Diagnostics
Genetic Counseling
Criminology/Forensics
Paternity testing
Archeology
Food testing
Evolutionary studies
Medicine
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
Vaccines :vare 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
Models of GD : GA to be vulnerable to a
disease so that the mechanism of the disease

can be investigated and potential therapeutic
agents tested.
Eg. mice GA with a cancer-predisposing
gene. Research to test the consequences of
exposure to various potential carcinogens and
to test the efficacy of preventive drugs.
Agriculture
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.
Spoilage of delicate products. Eg tomatoes if ripened are too

soft for shipping. In the past, tomatoes were harvested while
green and hard, refrigerated while shipping, and artificially
ripened in ethylene gas.
The softening of tomatoes is caused by the enzyme
polygalacturonase.
Now = The polygalacturonase gene has been inserted, in
reverse order, into tomato plants. This inactivates the original
enzyme-producing gene and produces a reduction in softening
of the tomatoes.
GA tomatoes can be shipped without expensive refrigeration,
which allows them to be produced more cheaply and sold at
lower price.
Agriculture
Environment. Many waste products of agriculture/industry do

not break down naturally/break down slowly. PROBLEM.
Many bacteria have been GE capable of breaking down oil and
other organic wastes
Cheese making industry : GE Saccharomyces cerevisiae able
to dispose of whey by converting lactose to alcohol.
Agricultural waste products, eg. corn husks, contain cellulose
that normally decomposes slowly. Can be converted into sugar
by cellulase. Cellulase has been inserted in E.coli making it
useful in waste management/disposal programs..

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Recombinant DNA Technology

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Recombinant DNA technology

be isolated, cut and spliced together

RDNA tech process

A gene is located on a chromosome map

enzymes cuts DNA

the host cell.

genetic material to other

enzymes. Restriction endonucleases

Specific palindromic sites

relatively small, many separate clones must be

Applications of recombinant DNA

The processes of inheritance and gene

Medicine production of industrial and commercial

Diagnostic kits Hep, AIDS

testing,Mapping the chromosomal location

Animals = models of genetic diseases Eg GE mice

determine a pathogen's surface configuration to a

disease so that the mechanism of the disease

Spoilage of delicate products. Eg tomatoes if ripened are too

Environment. Many waste products of agriculture/industry do

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