Recombinant DNA technology involves manipulating and combining DNA from different sources to produce new genetic combinations. This is done by using enzymes to cut DNA at specific locations and then join DNA fragments together. Vectors such as plasmids and bacteria are used to introduce the recombinant DNA into host cells, where it can be replicated. Key applications of this technology include producing therapeutic proteins like insulin, developing genetically engineered crops, and creating vaccines.

1. Basic Principle of
recombinant
DNA Technology
Dr. Sapna Saini
Asst. professor ( Ph’
biotechnology)
Chesta {15}
Priyanka {74}
B Pharmacy 6th

2. INTRODUCTION
• Recombinant DNA technology comprises altering genetic material outside an
organism to obtain enhanced and desired characteristics in living organisms or as
their products.
• This technology involves the insertion of DNA fragments from a variety of sources,
having a desirable gene sequence via appropriate vector.
• Recombinant DNA technology involves using enzymes and various laboratory
techniques to manipulate and isolate DNA segments of interest.
• This method can be used to combine (or splice) DNA from different species or to
create genes with new functions. The resulting copies are often referred to as
recombinant DNA.
• Such work typically involves propagating the recombinant DNA in a bacterial or
yeast cell, whose cellular machinery copies the engineered DNA along with its own.

4. CONTENT
S
1. Discovery
2. Goals and objectives
3. rDNA Technology procedures
4. Tools
5. Enzymes
6. Vectors
7. Host
8. Applications
9. Summary

5. DISCOVERY
The first recombinant DNA (rDNA) molecules were generated in
1973 by Paul Berg, Herbert Boyer, Annie Chang, and Stanley
Cohen of Stanford University and University of California San
Francisco. After preliminary experiments in 1973, the Cohen-
Boyer team was able to cut open a plasmid loop from one species
of bacteria, insert a gene from different bacterial species and
close the plasmid.
This created a recombinant DNA molecule-- a plasmid containing
recombined DNA from two different sources.

6. GOALS AND
OBJECTIVES
 Interpretation of hereditary diseases and related cures.
 Artificial synthesis of new genes.
 Modification of organisms' genome.
 Enhancement of the human genome.
 In agriculture, recombinant DNA has improved plant growth by
increasing nitrogen fixation efficiencies, by cloning bacterial genes,
and inserting them into plant cells.
 Other plants have been engineered to be resistant to caterpillar,
pests, and viruses by inserting resistant genes into plant genomes

7. PROCEDURES FOR RDNA
TECHNOLOGY
Steps in recombinant DNA technology:-
Recombinant DNA technology is performed according to specific processes, usually
involving the following steps:
 Isolating genetic material.
 Cutting DNA fragments at specific locations.
 Joining DNA fragments by ligation and homopolymer tailing.
 Inserting DNA into the host cell.
 Selecting and screening the transformed cells.

8. DIAGRAM

9. TOOLS USED

10. Enzymes used in recombinant DNA technology
 DNA ligase
 Reverse transcriptase
 Restriction endonuclease
 Terminal transcriptase
 Nuclease
 DNA polymerase
 Ribonuclease-H
 Alkaline phosphatase
 Polynucleotide kinase
ENZYMES

11.  1.DNA ligase:
 The enzyme DNA ligase joins the DNA fragments with cloning vector.
 2. Reverse transcriptase:
 RT is used to synthesize complementary strand (cDNA) from mRNA
template.
 It is also known as RNA dependent DNA polymerase
 3. Restriction endonuclease:
 Restriction endonuclease enzyme recognize and cut DNA strand at specific
sequence called restriction site.
 These enzyme is isolated from wide variety of microorganisms. Endonuclease
enzyme degrades foreign genome
 There are 3 types of restriction endonuclease:
 Type I Restriction endonuclease
 Type II Restriction endonuclease
 Type III Restriction endonuclease

12.  4. Terminal transferase:
 It is the enzyme that converts blunt end of DNA fragments into
sticky end.
 Terminal transferase enzyme synthesize short sequence of
complementary nucleotide at free ends of DNA, so that blunt end is
converted into sticky end.
 5. Nuclease:
 The enzyme nucleases hydrolyses the phosphodiester bond on DNA
strand creating 3’-OH group and 5’-P group.
 The gap is filled by DNA polymerase and strand is joined by DNA
ligase
 Nuclease are of two types; endonuclease and exonuclease
 6. DNA polymerase:
 DNA polymerase is a complex enzyme which synthesize nucleotide
complementary to template strand and help in elongation of strand
 It also helps to fill gap in double stranded DNA.

13.  7. Ribonuclease-H (RNase H):
 RNase-H removes mRNA from DNA-RNA heteroduplex and that
mRNA is used to synthesize cDNA
 It is isolated from retrovirus
 8. Alkaline phosphatase:
 The enzyme Alkaline phosphatase helps in removal of terminal
phosphate group from 5′ end
 It prevents self annealing of vector DNA soon after cut open by
restriction endonuclease
 9. Polynucleotide kinase:
 It adds phosphate group from ATP molecule to terminal 5’end after
dephosphorylation by alkaline phosphatase.

14. VECTORS
A vector, as related to molecular biology, is a DNA
molecule (often plasmid or virus) that is used as a
vehicle to carry a particular DNA segment into a host
cell as part of a cloning or recombinant DNA technique.
Plasmids and bacteriophages are the most common
vectors in recombinant DNA technology that are used
as they have a very high copy number.

15. PLASMIDS AS VECTOR
 Are defined as autonomous elements , whose
genomes exists in the cell as extra cellular
chromosomal units.
 Are self- replicating.
 The naturally occurring plasmids have been modified
to serve as vectors in the laboratories.
 Eg. pBR322, pUC vectors , Yeast plasmids etc.

16. BACTERIOPHAGE AS
VECTOR
 Are viruses that infect bacterial cells by
infecting their DNA into these cells.
 They are used as vectors coz they have a linear
DNA molecule.
 Eg. M13 , Lamda etc.
 Cosmids , Phagemids , BAC i.e; Bacterial Artificial
Chromosomes , YAC , MAC etc. are also used as
vectors for the process of recombinant DNA
technology.

17. PLASMID
BACTERIOPHAGE

18. HOST
For successful genetic engineering , a good Host is an
important device. Bacterium E. Coli is a very common host
for r-DNA technology.
Since isolation as well as cloning of DNA inserts in E. Coli
is very simple.
An ideal host organism is the one which is easy to
transform and in which the replication of r-DNA is simple.
E.Coli

19. APPLICATIONS
Recombinant DNA technology is used to produce artificial DNA by using a
combination of different genetic materials from various sources.
Applications and uses
i) Isolation, identification , mapping and sequencing of genes.
ii) Describing the function of a gene.
iii) Production of recombinant proteins for lab experiments.
iv) Development of drug delivery systems.
v) Recombinant Vaccines can be developed for prevention of many diseases
like Hepatitis-B.
vi) Synthesis of drugs, hormones and proteins. Insulin and growth
hormones are examples.
vii) Detection and Diagnosis of diseases like HIV.
viii) Treatment using gene silencing systems like CRISPR.

20. ix) Gene therapy which is used to correct the genetic defects.
x) Production of genetically modified crops in agriculture.
xi) Introduction of pest resistant genes in crops. For example
pest resistant cotton and pest resistant brinjal.
xii) Recombinant enzymes are used in the food industry for
example in production of cheese.
xiii)Transgenic animal production like rats, pigs etc.
Hence, we can conclude recombinant DNA technology is playing
a crucial role in various sectors like agriculture , food industry ,
vaccines development etc.

21. BIOSYNTHESIS OF
INTERFERONS
 Interferons are natural glycoproteins formed by Virus-infected
eukaryotic cells which protect host cells from virus infections.
 The substance was named interferon since it interfered with
development of viruses.
 These are manufactured by living animal cells, both in-vivo and
cultured cells.
 The substance in a virus that is responsible for interferon
synthesis by the host cell is known as Interferon Inducer. It is
normally a double-stranded RNA.
 The double-stranded RNA Virus such as Retrovirus can act as
interferon inducer deprived of replication.

23. BIOSYNTHESIS OF VACCINE i.e,
HEPATITIS
 Recombinant vaccines are made using bacterial or yeast
cells to manufacture the vaccine. A small piece of DNA
is taken from the virus or bacterium against which we
want to protect and inserted into the manufacturing
cells.
 Hepatitis B vaccine recombinant contains a non infectious
hepatitis B viral antigen, HBsAg, which is produced in
yeast cell using recombinant DNA technology. Hepatitis
B vaccine is a whitish sterile suspension in prefill
injection device, packed in aluminium foil pouch, and vial.

25. HUMULIN
PRODUCTION
Recombinant Insulin is the outcome of effective genetic
engineering. In early days, production of insulin was
hectic as two chains viz, Chain A and Chain B were
formulated then they were connected via chemically to
yield insulin.
In this case, genes coding for human insulin are inserted
into bacteria. Bacteria produce insulin, which is harvested
and used as the active ingredient in Humulin. Humulin N is
formulated to have a slower onset of action than regular
insulin and a longer duration of activity (slightly less than
24 hours).

27. SUMMARY
 Recombinant DNA technology is an extremely important research tool in biology. It allows
scientists to manipulate DNA fragments in order to study them in the lab. It involves using a
variety of laboratory methods to put a piece of DNA into a bacterial or yeast cell.
 Recombinant DNA technology comprises altering genetic material outside an organism to obtain
enhanced and desired characteristics in living organisms or as their products. This technology
involves the insertion of DNA fragments from a variety of sources, having a desirable gene
sequence via appropriate vector .
 Recombinant DNA technology has also proven important to the production of vaccines and protein
therapies such as human insulin, interferon and human growth hormone. It is also used to produce
clotting factors for treating haemophilia and in the development of gene therapy.
 One of the key techniques used in biotechnology is genetic engineering, which allows scientists to
modify the genetic makeup of organisms to achieve desired outcomes. This can involve inserting
genes from one organism into another, creating new traits or modifying existing ones.

28. THANKYOU