2022-23

TOPIC: Recombinant DNA Technology

Submitted by: Abhinav Choudhary

Class: XII
 Certificate

This is to certify that the project work titled ………………………………………….

……………………………………………………….............is record of original work

done by ………….…… with registration number …………... under my
supervision and guidance.

The Principal Teacher in charge

Submitted for Practical Examination held on ____________________

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 ACKNOWLEDGEMENT
Primarily I would thank God for being able to
complete this project with success. Then I would like to thank
my biology teacher Mrs. Akanksha Dubey, whose valuable
guidance has been the ones that helped me patch this project
and make it full proof success her suggestions and
instructions has served as the major contributor towards the
completion of the project.
Then I would like to thank my parents and friends
who has helped me with their valuable suggestion and
guidance has been helpful in various phases of the
completion of the project.
Last but not least I would like to thank my
classmates who helped me a lot.

Abhinav Choudhary
Class – 12th Biology
 INDEX

1.Introduction
2.History
3.Basic principle of recombinant DNA
4.Important tools for genetic
engineering/recombinant technology
5.Processes of recombinant DNA
technology
6.Obtaining foreign gene product
7.Applications of recombinant DNA
technology
8.Conclusion
 Introduction:-

Definition: It is a technology of joining together of DNA

molecules from two different species that are inserted into a
host organism to produce new genetic combinations that are
of value to science, medicine, agriculture, and industry.

 Since the focus of all genetics is the gene, the fundamental goal of
laboratory geneticists is to isolate, characterize, and manipulate genes.
 Although it is relatively easy to isolate a sample of DNA
 Consider the fact that each cell contains approximately 2 meters (6 feet)
of DNA.
 Therefore, a small tissue sample will contain many kilometers of DNA.
 However, recombinant DNA technology has made it possible to isolate
one gene or any other segment of DNA, enabling researchers to
determine its nucleotide sequence, study its transcripts, mutate it in
highly specific ways, and reinsert the modified sequence into a living
organism.
 HISTORY:-
History of Genetic Engineering/Recombinant DNA
technology

In conjunction with his studies of the tumor

virus SV40, in 1972, Paul Berg succeeded in
inserting DNA from a bacterium into the
virus' DNA. He thereby created the first DNA
molecule made of parts from different
organisms. This type of molecule became
known as "hybrid DNA" or "recombinant
DNA". Among other things, Paul Berg's
method opened the way to creating bacteria
that produce substances used in medicines.

Paul Berg is the "father of genetic

engineering".
History of Recombinant DNA technology
In 1973, Herbert Boyer, of the University of California at San
Francisco, and Stanley Cohen, at Stanford University, reported
the construction of functional organisms that combined and
replicated genetic information from different species. Their
experiments dramatically demonstrated the potential impact of
DNA recombinant engineering on medicine and pharmacology,
industry and agriculture.

Boyer and Cohen's achievement represented an advance upon the

ingenious techniques developed by Paul Berg, in 1972, for inserting
viral DNA into bacterial DNA. It was a creative synthesis of earlier
research that made use of:
✓Living organisms able to serve as carriers for genes from
another organism.
✓Enzymes to cleave and rejoin DNA fragments that contain
such genes.
✓DNA molecules from one organism precisely targeted and
manipulated for insertion into the DNA of another organism.
Basic Principle of Recombinant DNA
Technology :
The DNA is
inserted into
another DNA
molecule called
vector.

The recombinant
vector is then
introduced into a
host cell
where it
replicates itself
and multiple
copy of gene
produced.
Recombinant DNA
Technologies
1. Gene of interest
(DNA) is isolated
(DNA fragment)

2. A desired gene is
inserted into a DNA
molecule - vector
(plasmid, bacteriophage or a
viral genome)
3. After entering the host
cell, vector
grown/relpicate to form
a clone.
(bacteria, yeast, plant or animal
cell)

4. Large quantities of the gene product can be harvested

from the clone.
 Important Tools For Genetic
engineering/recombinant technology :-
Restriction Enzymes
Naturally produced by bacteria – Restriction
Endonucleases
Natural function - destroy bacteriophage DNA in bacterial cells
Cannot digest host DNA with methylated C (cytosine)

Restriction Endonucleases (RE): Endonuclease are enzymes that

produce internal cut called cleavage in DNA molecules. A class of
endonucleases that cleaves/cut DNA only within or near those sites
which have specific base sequences, such endonucleases are known as
restriction endonucleases or restriction enzymes and site recognised by
them are called recognition sequences or recognition sites. There are
three types of RE.
Type I Restriction Endonuclease
Type II Restriction Endonuclease
Type III Restriction Endonuclease

Type II RE
–They recognize a specific nucleotide sequence (recognition site) and
cut a DNA molecule within that sequence (breaks down the bond between
two nucleotides).
–Most of the enzymes recognize hexanucleotide (6bp) sequence but
enzymes with 4, 5 and 8 bp recognition site are also present.
–Some enzymes produced blunt ends while some produce 5’ or 3’ staggered
sticky ends.
sticky ends blunt ends

Selected Restriction enzymes used

in rDNA technology
 How RE works in Recombinant DNA
Technology?

Ligase
• DNA ligase is a enzyme that can link together DNA strands that have
double-strand breaks (a break in both complementary strands of DNA).
- Naturally DNA ligase has applications in both DNA replication and
DNA repair.
-Needs ATP

• DNA ligase has extensive use in molecular biology laboratories for

genetic recombination experiments
 Vectors
“A vector is a DNA molecule that has the ability to replicate autonomously in
an appropriate host cells and serve as a vehicle that carry DNA fragment or
insert to be cloned.” Therefore, a vector must have an origin of DNA
replication (ori) that functions in the host cell. Any extra-chromosomal small
genome eg. Plasmid, Phage or virus may be used as a vector.

ATP
 Type of Vectors

•Plasmid vector • Bacteriophage vector • Cosmid vector • Phagemid vector

• Phasmid vector
•BAC • YAC • PAC
Plasmid Vector
• Many different E. coli plasmids are used as vectors. The natural plasmid have
been modified, shortened, reconstructed and recombined both in vitro and in
vivo to create plasmid of enhance utility and specific functions. • Examples:
pBR322, pUC18/19,
pGEM3Z, pTZ57R/T

pBR322:One of the first widely used E. coli cloning vectors

 Hosts for DNA Recombinant
Technology
1. Bacteria
- E. coli - used because is easily grown and its genomics are well
understood. Gene product is purified from host cells
2. Yeasts - Saccharomyces cerevisiae
 Used because it is easily grown and its genomics are known
 May express eukaryotic genes easily
 Easily collected and purified
3. Plant cells and whole plants
 May express eukaryotic genes easily
 Plants are easily grown - produce plants with new properties.
4. Mammalian cells
 May express eukaryotic
genes easily
Harder to grow
Medical use.
5. Fish Cell Lines
 • Isolation of DNA
Processes of • Cutting of DNA at specific location
Recombinant • Amplification of gene of intrest using PCR
DNA
Technolog • Insertion of Recombinant DNA into Host
• Obtaining the foreign gene product
Has five steps

Isolation of DNA
DNA isolation is a process
of purification of DNA from
sample using a combination of
physical and chemical
methods. The basic steps in a
DNA extraction are as below:
• Cells which are to be
studied need to be
collected.
• Breaking the cell
membranes to expose the
DNA along with the
cytoplasm - Cell Lysis.
• Lipids from the cell
membrane and the nuclear
membranes are broken
down with detergents.
• Breaking proteins by adding an Proteinase
• After centrifugation of the sample, denaturated proteins stay in the
organic phase while aqueous phase
containing nucleic acid

Cut DNA with restriction

enzymes

restriction
enzyme

Restriction enzymes (endonuclease) recognize specific

bases pair sequences in DNA called restriction sites
and cleave the DNA by hydrolyzing the
phosphodiester bond.
✓ Restriction fragment ends have 5’ phosphates & 3’
hydroxyls.
 Step 3: Amplification of gene of interest using
PCR

Polymerase
chain reaction
– It is a
process to
amplify the
gene once the
proper gene of
interest has
been cut using
the restriction
enzymes.
30-35 cycles
 Insertion of
DNA into the
Host/vector.
• By digesting both the plasmid
and DNA with the same
restriction enzyme we can
create thousands of DNA
fragments, one fragment with
the gene that we want, and with
compatible sticky ends on
bacterial plasmids.
• After mixing, the desired
fragments and cut plasmids form
complementary pairs that are
then joined by DNA ligase.
• This creates a mixture of
recombinant DNA molecules.

Obtaining the foreign gene product

 Applications of
Recombinant DNA
Technology
1
. Scientific applications
.

 Many copies of DNA can be produced

 Increase understanding of DNA
 Identify mutations in DNA
 Alter the phenotype of an organism
2. Diagnose genetic disease
3. Recombinant DNA techniques can be used to for genetic fingerprinting
identification
▪ DNA fingerprinting to identify the source of bacterial or viral pathogens.
▪ bioterrorism attacks (Anthrax in U.S. Mail)
▪ medical negligence (Tracing HIV to a physician who injected it)
▪ outbreaks of foodborne diseases
4. Agricultural Applications
Cells from plants with desirable characteristics can be cloned to produce many identical cells, then
can be used to produce whole plants from which seeds can be harvested.
5. Nanotechnology
 Bacteria can make molecule-sized particles
 Bacillus cells growing on selenium form chains of elemental selenium

6. Therapeutic Applications
 Produce human proteins – hormones and enzymes (Insulin, hGH, INFα, INFβ and INFγ)
 Vaccines : Cells and viruses can be modified to produce a pathogen’s surface protein (Influenza;
Hepatitis B;
Cervical
cancer
vaccine)
 Conclusion

1.Recombinant DNA technology is not only an

important tool in scientific research, but has also
resulted in enormous progress in the diagnosis and
treatment of certain diseases and genetic disorders
in many areas of medicine

2.Thus Recombinant DNA technology is beginning

to significantly alter the practice of medicine by
providing new diagnostic and therapeutic agents
and revealing molecular mechanisms of disease.