2. Contents….
• What is recombinant DNA technology.
• Biological and chemical tools used in rDNA-
technology.
• Procedure.
• PCR technique.
• Applications.
• Conclusion.
3. Recombinant DNA Technology
Production of a unique DNA molecule by joining
together two or more DNA fragments not normally
associated with each other, which can replicate in
the living cell.
Recombinant DNA is also called Chimeric DNA
Developed by Boyer and Cohen in 1973
7. ENZYMES USED IN R-DNA TECH
• Enzymes used in R-DNA technology falls into 4 broad
categories :
• 1) Template dependent DNA polymerase : those which are
used in synthesis of new polynucleotide complementary to
an existing DNA or RNA template. Example: DNA polymeraseI,
DNA poly III, Reverse transcriptase, Taq polymerase etc .
• 2) Nucleases : these enzymes used to degrade DNA molecules
by breaking phosphodiester bonds. Example : exonucleases
and endonucleases . Endonucleases also have a broad
category i.e restriction endonucleases, used to cut DNA at
specific sequence.
8. Continued….
• 3) End modification enzymes : these enzymes make
changes to the ends of DNA molecules. Example :
Terminal deoxynucleotidyl transferase , Alkaline
phosphatase , T4 polynucleotide kinase.
• 4) Ligases : DNA ligases join DNA molecules
together by synthesizing phosphodiester bonds
between nucleotides at the ends or between two
different molecules. They can be Linkers and
Adaptors.
9. Restriction Endonucleases
Important tool for rDNA technology is the Restriction Enzyme
Bacterial enzymes that cut DNA molecules only at restriction sites
Molecular scissors
Palindromic sequences are the recognition sites
eg: EcoRI with recognition site GAATTC
5´ GAATTC 3´
3´ CTTAAG 5
Categorized into two groups based on type of cut
• Cuts with sticky ends
• Cuts with blunt ends
if one strand extends beyond the complementary region, then the
DNA is said to possess an overhang and it will have sticky ends.
12. Isolation of gene
DNA molecule is extracted from the cell by using cell lysing method
Homogenization
Centrifugation
Gene of interest is isolated using probes and electrophoresis
DNA , RNA and Plasmids can be isolated by different methods .
DNA which is to be cloned have to be inserted in to a vector
molecule which act as a carrier of the DNA to the host cell.
13. Vectors
• Vector is an autonomously replicating genetic
element used to carry a fragment of target DNA into
a host cell for the purpose of clonning and
expression.
• Vectors should be :
• 1) able to replicate inside host
• 2)should contain genetic marker to select for host
cell containing vector
• 3) should have unique restriction enzyme sites for
insertional cloning.
16. HOST (in vivo gene amplification or
expression)
• The vector carrying desired gene should be inserted
into a suitable host.
• Host should be suitable enough to provide the
vector a shelter, as well as freedom to replicate
inside it.
• The host enzymes shouldn’t interfere with the
external genetic material.
• Among prokaryotes E.coli is suitable host used in
RDNA TECH.
• Among eukaryotes yeast is suitable.
17. Cloning-Transformation:
• It is introduced into host cell by adding it into
culture of plasmid free bacteria or animal
cells.
• Heating and adding calcium chloride favors
the transformation
• Once inside the host cell, the recombinant
DNA begins to multiply and form the desired
product.
18. Overview of rDNA technology
Bacterial cell
DNA containing
gene of interest
Bacterial
chromosome Plasmid
Isolate Plasmid
Gene of interest
Enzymatically cleave
DNA into fragments.
Isolate fragment with the
gene of interest.
Insert gene into plasmid.
Insert plasmid and gene
into bacterium.
Culture bacteria.
20. Selection of recombinant cells
• Only bacteria which have
taken up plasmid grow on
amphicillin.
• Blue-white selection (x-gal
medium) :
– white colonies have insert
– blue colonies have no
insert
• The transformed cell are cultured
and multiplied.
• Colony of cell each containing the
copy of the recombinant plasmid
is obtained.
21. Non-Bacterial transformation
Microinjection, using micropipette.
The host cells are bombarded with high
velocity micro-projectiles, such as particles
of gold or tungsten that have been coated
with DNA.
• Phage introduction-Phage is used instead
of bacteria.
• In vitro packaging of a vector is used.
• lambda or MI3 phages to produce phage
plaques which contain recombinantsIt
• Electroporation-involves applying a brief
(milliseconds) pulse high voltage electricity
to create tiny holes in the bacterial cell wall
that allows DNA to enter.
22. PCR TECHNIQUE
(polymerase chain reaction)
This technique used in desired gene amplification.
This is a rapid and versatile in vitro method for amplification
of target DNA sequences.
This technique was developed by Kerry mullis (1985).
This method includes
1) primer design
2) degenerate primers
3) reaction cycle
* denaturation (93-95 c)
* primer annealing (50-70 c)
* DNA synthesis (70-75 c)
25. Applications of recombinant DNA
technology
•
Recombinant DNA is widely used
in biotechnology, medicine and research. Today, recombinant proteins
and other products that result from the use of rDNA technology
• The most common application of recombinant DNA is in basic research, in
the biological and biomedical sciences, Recombinant DNA is used to
identify, map and sequence genes, and to determine their function. rDNA
probes are employed in analyzing gene expression within individual cells,
and throughout the tissues of whole organisms. Recombinant proteins are
widely used to generate antibody probes for examining protein synthesis
within cells and organisms.
26. • Many additional practical applications of recombinant DNA are found
in industry, food production, human and veterinary medicine,
agriculture, and bioengineering.
• Recombinant chymosinFound in rennet, is an enzyme required to
manufacture cheese. It was the first genetically engineered food
additive used commercially. Traditionally, processors obtained
chymosin from rennet, a preparation derived from the fourth stomach
of milk-fed calves. Scientists engineered a non-pathogenic strain (K-
12) of E. coli bacteria for large-scale laboratory production of the
enzyme. This microbiologically produced recombinant enzyme,
identical structurally to the calf derived enzyme, costs less and is
produced in abundant quantities. Today about 60% of U.S. hard
cheese is made with genetically engineered chymosin.
27. • Recombinant human insulin Almost completely replaced
insulin obtained from animal sources (e.g. pigs and cattle)
for the treatment of insulin-dependent diabetes. A variety of
different recombinant insulin preparations are in widespread
use. Recombinant insulin is synthesized by inserting the
human insulin gene into E. coli, which then produces insulin
for human use.Recombinant human growth hormone (HGH,
somatotropin) Administered to patients whose pituitary
glands generate insufficient quantities to support normal
growth and development.
28. • Recombinant blood clotting factor VIII A blood-clotting protein
that is administered to patients with forms of the bleeding
disorder hemophilia, who are unable to produce factor VIII in
quantities sufficient to support normal blood coagulation. Before
the development of recombinant factor VIII, the protein was
obtained by processing large quantities of human blood from
multiple donors, which carried a very high risk of transmission
of blood borne infectious diseases, for example HIV and
hepatitis B. DrugBank entry Recombinant hepatitis B
vaccine Hepatitis B infection is controlled through the use of a
recombinant hepatitis B vaccine, which contains a form of the
hepatitis B virus surface antigen that is produced in yeast cells.
29. • The development of the recombinant subunit vaccine was an
important and necessary development because hepatitis B
virus, unlike other common viruses such as polio virus, cannot
be grown in vitro. Vaccine information from Hepatitis B
FoundationDiagnosis of infection with HIV Each of the three
widely used methods for diagnosing HIV infection has been
developed using recombinant DNA. The antibody test
(ELISA or western blot) uses a recombinant HIV protein to test
for the presence of antibodies that the body has produced in
response to an HIV infection. The DNA test looks for the
presence of HIV genetic material using reverse transcriptase
polymerase chain reaction (RT-PCR). Development of the RT-
PCR test was made possible by the molecular cloning and
sequence analysis of HIV genomes .
30. • GOLDEN rice A recombinant variety of rice that has been engineered to
express the enzymes responsible for β-carotene biosynthesis. This variety of
rice holds substantial promise for reducing the incidence of vitamin A
deficiency in the world's population. Golden rice is not currently in use,
pending the resolution of regulatory issues. Herbicide-resistant
crops Commercial varieties of important agricultural crops (including soy,
maize/corn, sorghum, canola, alfalfa and cotton) have been developed that
incorporate a recombinant gene that results in resistance to the
herbicide glyphosate (trade name Roundup), and simplifies weed control by
glyphosate application.These crops are in common commercial use in several
countries. Insect-resistant crops Bacillus thuringeiensis is a bacterium that
naturally produces a protein (Bt toxin) with insecticidal properties.[The
bacterium has been applied to crops as an insect-control strategy for many
years, and this practice has been widely adopted in agriculture and gardening.
Recently, plants have been developed that express a recombinant form of the
bacterial protein, which may effectively control some insect predators.
31. Applications…
Pharmaceutical and Therapeutic Applications
Gene therapy
Medical diagnosis
Xenotransplants
Agricultural Applications
Production of transgenic organisms
32. Environmental applications
• Many waste products of agriculture/industry do not break
down naturally/break down slowly.
Many bacteria have been GE capable of breaking down oil and
other organic wastes in 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..
33. Conclusion…
• R-DNA technology is the new era in field of
biology which has huge applications and
possibilities.
• It is now a great topic for researching and
exploring .
• It can take us to a new level of biological
science , which is “biotechnology” – living
organisms manipulated by technology .
