rDNA used in production of new technology

RECOMBINENT DNA TECHNOLOGY –
PRINCIPLE,STEPS AND APPLICATIONS.
Presented by,
EDEN FIONA MATHIAS
1st
M. Pharm
(Department of Pharmacology)
1

 Introduction
 Discovery
 Goals and objectives
 Principle
 rDNA technology procedure
 Enzymes
 Applications
 Conclusion
 Reference
•
Contents
2

Recombinant DNA technology
 Recombinant DNA technology, which is also called gene cloning or molecular
cloning, is a general term that encompasses a number of experimental protocols
leading to the transfer of genetic information (DNA) from one organism to another.
 In simple words, rDNA technology involves joining together of DNA molecules from
two different species.
3
Fig: 1 Recombinant DNA Technology

Discovery of Recombinant DNA Technology
 The discovery of Recombinant DNA in 1973 by Herbert
Boyer and his colleague Stanley N. Cohen at Stanford
University Medical School is the single greatest
breakthrough in medical biotechnology.
 Herbert Boyer and his colleague used molecular cloning to
create recombinant DNA from the bacterial genomes; DNA
and plasmid (Russell & Sambrook, 2001).
4
Fig : 2

 They transformed E. Coli by inserting foreign DNA segments into the bacterial DNA
strand to create a new genome that could express the bacterial genes contained in its
original DNA, as well as genes in the foreign DNA insert from the donor organism.
 As a result, the transformed bacteria commonly referred to as the recombinant cell
was able to express replicate the foreign DNA insert through bacterial division to
produce more transformants. In addition, the recombinant bacteria produced
respective proteins encoded by the genes in the foreign DNA insert.
5

Goals of rDNA Technology
 To isolate and characterize a gene.
 To make desired alteration in one or more isolated genes.
 To return altered genes to living cells.
 Artificially synthesize new gene.
 Alternating the genome of an organism.
 Understanding the hereditary diseases and their cure.
 Improving the human genome.
6

Principle Involved In rDNA Technology
 The DNA from the donor organism is extracted, enzymatically cleaved and joined to
another DNA entity (a cloning vector).
 Forms a new, recombined DNA molecule (cloning vector- insert DNA construct).
 Transferred into and maintained within a host cell. The introduction of DNA into a
bacterial host cell is called transformation.
 Host cells that take up the DNA construct are identified and selected.
 The protein product encoded by the cloned DNA sequence is produced in the host cell.
7

Procedure Involved In rDNA Technology
8
Fig : 3 showing Diagrammatic representation of rDNA technology

A. ISOLATION OF DNA
B. CUTTING OF DNA
C. JOINING OF DNA
D. AMPLIFICATION OF DNA
E . TRANSFORMATION OF
DNA
9

A. ISOLATION OF DNA
10
Cellular lysis
Detergent / Lysosomes
Chelating Agents
EDTA / Citrate
Proteinase Agents
Proteinase K
Phenol Extraction
Phenol / Chloroform
Alcohol Precipitation
70%/100%Ethanol
Redissolve DNA
TE Buffer (Tris-EDTA)
Fig : 4 showing Extraction of DNA

11
B. CUTTING OF DNA
 Restriction enzyme digestion are performed by incubating purified DNA molecules with
Restriction enzymes, at the optimal conditions for that specific enzymes.
 Incubate the reaction mixture at the optimal temperature for the enzyme (usually around
37 ) for a specified duration (30minutes to several hours), allowing the enzyme to cut
℃
the DNA.
 Stopping the Reaction: The reaction can be stopped by heating (inactivating the
enzyme ) or adding EDTA, which chelates divalent cations required for enzyme activity.

 Both the purified DNA and the vector DNA will be cut at the palindromic site
(palindromic sites are DNA sequences that read the same backward as forward, often
recognized by restriction enzymes).
 Agarose gel electrophoresis is employed to check the progression of RE digestion.
 DNA is negatively charged molecule,hence it moves towards the positive electrode
(anode).
 The process is repeated with the vector DNA also.
12
Fig : 5 showing Agarose gel electrophoresis

Fig : 6 showing cutting of DNA
13

VECTORS USED IN DNA TECHNOLOGY
 A vector is an area of DNA that can join another DNA part without losing the limit for
self- replication.
 Features:
 Should be capable of replicating in host cell
 Should have convenient RE sites for inserting DNA of interest.
 Should be small and easy to isolate.
14

TYPES OF VECTORS USED:
PLASMID
 Self-replicating, double stranded, circular DNA molecules. Maintained in bacteria as
independent extrachromosomal entities. Ranges in size from less than 1 kb to more
than 500 kb. Plasmid has a sequence that functions as an origin of DNA replication,
without this site, it cannot replicate in a host cell.
15
Fig : 7 showing plasmid cloning vector

LAMBDA PHAGE:
 Virus which infects the bacteria are called phages. They have a linear DNA molecule.
Carry DNA fragments upto 10-20kbp.
16
Fig : 8 showing lamda phage vector

COSMIID:
• They are plasmids which combine the features of plasmid and phages. They contain
special genes called cos site (needed for packing lambda DNA into phage particles).
Carry DNA fragments up to 30-50 kbp.
17
Fig : 9 showing cosmid vector

 The joining of DNA involves several
processes.
 After having cut the source DNA as well as the
vector DNA with a specific restriction
enzyme,the cut out ‘gene of interest’ from the
source DNA and the cut vector with space are
mixed and ligase is added.
 This results in the preparation of recombinant
DNA.
C. CLONING OF FOREIGN DNA INTO PLASMID VECTOR.
18
Fig : 10 showing joining of DNA

 PCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the
gene (or DNA) of interest is synthesised in vitro using two sets of primers (small
chemically synthesised oligonucleotides that are complementary to the regions of
DNA) and the enzyme DNA polymerase.
D. AMPLIFICATION OF GENE OF INTEREST USING PCCR
19

Fig : 11 showing polymerase chain reaction
20

 The enzyme extents the primers using the nucleotides provided in the reaction and the
genome DNA as template.
 If the process of replication of DNA is repeated many times, i.e., 1 billions copies are
made.
 Such repeated amplification is achieved by the use of a thermostable DNA polymerase
(isolated from a bacterium, Thermus aquaticus), which remain active during the high
temperature induced denaturation of double stranded DNA.
 The amplified fragment if desired can now be used to ligate with a vector for further
cloning.
21

E. TRANSFORMATION
 The next step in a recombinant DNA experiment requires the uptake of the cloned
plasmid DNA by a bacterial cell, usually E. coli
 The process of introducing purified DNA into a bacterial cell is called transformation,
and a cell that is capable of taking up DNA is said to be competent. Competence
occurs naturally in many bacteria.
 However, competence can be induced by various special treatments, such as cold
calcium chloride.
22

 A brief heat shock facilitates the uptake of exogenous DNA molecules. Two parameters-
transformation frequency and transformation efficiency are used to assess the success of
DNA transformation.
 After the transformation step, it is necessary to identify those cells that contain plasmids
with cloned DNA.
 Following transformation, the cells are incubated in medium without antibiotics to allow
the antibiotic resistance genes to be expressed, and then the transformation mixture is
plated onto medium that contains the antibiotic ampicillin.
23

Fig : 12 Transformation of rDNA into the bacterial host cells
24

ENZYMES USED IN RECOMBINANT DNA TECHNOLOGY
Type II restriction endonuclease
DNA ligase
Reverse transcriptase
DNA polymerase I
Polynucleotide Kinase
Terminal transferase
Exonuclease III
Bacteriophage (lamda) exonuclease
Alkaline phosphatase
• Bind to DNA molecules
• Cleaves DNA at specific sites
• Make a DNA copy of RNA molecule
• Full single stranded gapes of DNA duplex
• Adds a phosephate to the 5’-OH end of a polynucleotide
• Adds homopolymer tails to the 3’-OH ends
• Removes nucleotide residues from the the 3’ ends
• Removes nucleotides from the 5’ ends
• Removes terminal phosphates
25

26
APPLICATIONS OF rDNA TECHNOLOGY
 Agriculture - Recombinant DNA technology is used in agriculture to modify crops.
Proven beneficial in increasing crop yield, enhancing resistance to pests, and
promoting the growth and development of given plants.
 Medicine- In medicine, recombinant DNA technology is used for the production of
various antibiotics, hormones, interferon, and vaccines, etc.
 For instance, using E. coli bacteria as host cells, insulin is one of the most commonly
produced hormones through recombinant DNA technology.

27
Fig : 13 showing Genetically engineered insulin

 Gene therapy involves the introduction of given genes into the genome of an
individual in order to repair mutations. Viruses are intracellular parasites and so they
have been used to introduce genes into the cell for gene therapy.
28
Fig : 14 showing gene therapy

 Development of vaccines and cancer treatment - Today, cancer is one of the leading
causes of death. Using recombinant DNA technology, it will be possible to treat these
diseases effective genes.
 Development of transgenic animals- Ex: Glofish are a type of zebrafish with
recombinant DNA, Genes for fluorescent proteins have been inserted into their
genome to produce their fluorescent colours.
29

30
CONCLUSION
 Recombinant DNA (rDNA) technology has revolutionized biotechnology and medicine
by enabling the manipulation of genetic material.
 Its applications range from the production of insulin and vaccines to advancements in
agriculture through genetically modified organisms (GMOs).
 While rDNA technology holds immense potential for solving global challenges, such as
food security and disease management, it also raises ethical and safety considerations
that must be carefully addressed.
 Overall, rDNA technology is a powerful tool that continues to shape the future of
science and health.

31
REFERENCE:
1. Satyanarayana U. Biotechnology. Kolkata: Books and Allied Pvt Ltd .;2005;pp:
75-83.
2. Gerdts V, Van Drunen Littel-van den Hurk S. Applications of recombinant DNA
technology in vaccine development. Expert Rev Vaccines. 2008;7(1):77-93.
Available from: https://doi.org/10.1586/14760584.7.1.77. [Accessed on 12th
october 2024].
3. Hargreaves A, Gokhman I. Overview of recombinant DNA technology and its
applications. Biotechnology Advancement. 2011;29(5):700-12. Available from:
https://doi.org/10.1016/j.biotechadv.2011.05.009. [Accessed on 12th october
2024].

3. Walsh G. Pharmaceutical Biotechnology: concepts and applications. New Delhi: CBS
Distributors Pvt Ltd; 2011; pp:
4. Ghosh R. Recombinant DNA technology: principles and applications. Journal on
Genetic Engineering Biotechnology.2014;12(2):165-76. Available from:
https://doi.org/10.1016/j.jgeb.2014.04.001. [Accessed on 12th october 2024].
5. Jogdand S.N. Gene Biotechnology. 4th ed. Himalaya Publishing House; 2016; pp:
32

rDNA used in production of new technology

More Related Content

Similar to rDNA used in production of new technology

More from gaviinpereira2603

Recently uploaded

rDNA used in production of new technology