This presentation consist of Basic Overview of recombinent DNA Technology/ Genetic Engineering

1. Recombinant DNA Technology
Rajesh Kumar Singh, Ph.D.
Division of Genetics,
IARI, New Delhi

2. Introduction
 Recombinant DNA Technology (also Know as
Genetic Engineering) is the set of
techniques that involves the identification,
isolation and insertion of Gene of interest in
to a vector to form a recombinant DNA
molecule and production of large quantities'
of that gene fragment or product encoded
by that gene.

3. History
 Paul Berg, Herbert W. Boyer and stanly N. Cohen were
the pioneer in the field of Recombinant DNA Technology
but other Scientis also made important contributions as
well.
 In 1970 Werner Arber and Hamilton O discovered
Restriction enzymes.
 In 1972,Paulberg developed the First Recombinant DNA
molecule that combine DNA from SV40 virus and lambda
page.
 1973, Together with Stanley Cohen, Boyer demonstrated
the possibility of producing recombinant DNA in
bacteria.

4. Steps in rDNA technology
 Step 1: Identification & Isolation of Genes of Interest or
DNA fragments to be cloned.
 Step 2:Instertion of isolated genes in a suitable Vector.
 Step 3: Introduction of this vector in to suitable
organism/ cell called host ( Transformation).
 Step 4: Selection of transformed host cell.
 Step 5: Multiplication or expression of the introduced
gene in the host cell.

5. Requirements
 Target DNA/ Gene of interest:
 Enzymes
 Vector
 Host

6. Identification & Isolation of Gene of
Interest
From where we get this gene of intrest?
 Genomic Library
 cDNA Library
 Chemical synthesis of gene if we
know the sequence
 If the no of copies of the desired
gene is not enough for gene cloning
we can opt for gene amplification
technique like PCR

7. Enzymes for DNA manipulation
 Template –dependent DNA polymerase :
 DNA Polymerases I
 Taq DNA Polymerase
 Reverse Transcriptase
 Nucleases
 Mugbean nuclease
 S1Nuclease
 Rnase A
 Rnase H
 Restriction Endonuclease
 Type I
 Type II
 Type III
 Type IIs
 End-modification Enztymes
 Terminal deoxynucleotidyl tansferease
 Alkaline phosphatase
 T4 poly nucleotudw kinase
 Ligases

8. Vectors: The DNA molecules that as transporting vehicle
which carries foreign DNA into a host cell for the purpose of
cloning and expression
 Important Properties of a Vector:
 Ability to replicate on host cells
 MCS (multiple cloning sites) with unique restriction sites.
 Genetic Marker to select host cells containing Recombinant DNA.
 Low molecular weight.
 Vectors for E.Coli: Plasmid vectors (pBR 322, pUC 19 etc)
Viral DNA based Vectors Cosmid s
 Vectors for Yeast: YEp, YIp, YRp, YCp, YAC
 Vectors for Animals: P element for insects, Viral DNA based
vectors
 Vectors for plant: Plasmid Based & Viral vectors

9. Maximum DNA insert possible
with different cloning vectors
Vector Host Insert Size
M13 E. Coli 1-4 kb
Plasmid E. Coli 1-5 kb
Lambda Phase E. Coli 5-25 kb
Cosmids E. Coli 35-45 kb
P1 phase E. Coli 70-100 kb
PACs E. Coli 100-300 kb
BACs E. Coli <300 kb
YACs S. Cerevisiae 200-2000 kb
Source: Principle of Gene manipulation by S. B. Primrose

10. Gene Transformation Methods
There are multiple ways to deliberately introduce
foreign DNA or RNA into bacteria or eukaryotic cells
in molecular biology and scientific research.
 Transformation
 Transduction
 Conjugation
 Transfection
 Chemical methods (Cacl2 Mediated, PEG Mediated,
Calcium Phosphate, Liposome and Lipofection,
Protoplast Fusion, Receptor Mediated)
 Physical methods (Liposome encapsulation,
Microinjection, Particle bombardment,
Electroporation)

11. Selection of Transformed
Cells
 Genetic selection of transformed or transfected cells is
a significant step of Recombinant DNA technology and is
achieved by two ways:
 Selectable Markers: Gene encode a product that allows
the artificial selections of transformed cells.
 Positive selectable marker genes
 Negative Marker Genes
 Scorable/ screenable markers: also called as visual
marker, gene, generate a product that can be detected
using a simple and often quantitative assay

12. Expression Vectors and Expression
Systems
 The vectors designed for production of a protein
specified by the DNA insert is an Expression Vector
 A system in which a cloned gene can be expressed is an
expression system
 Prokaryotic (E. coli, Bacillus subtilis, Staphylococcus
carnosus, Streptomyces lividans)
 Eukaryotic (Yeast, Aspergillus niger, Baculovirus-Insect
Cells, Mammalian Cells (e.g. Chinese Hamster Ovary cells)

13. Recombinant DNA Technology

14. Applications of Recombinant DNA
technology
Source: Khan, Suliman, et al. ." International journal of genomics (2016).

15. Limitations of Recombinant
DNA technology
•Destruction of native species in the environment the genetically
modified species are introduced in.
•Resilient plants can theoretically give rise to resilient weeds which
can be difficult to control.
•Cross contamination and migration of proprietary DNA between
organisms.
•Recombinant organisms contaminating the natural environment.
•Creation of superbug is hypothesized.
•Ethical concern about humans trying to play God and mess with
the nature’s way of selection. It is exaggerated by the fear of
unknown of what all can be created using the technology and how
is it going to impact the civilization.
•Such a system might lead to people having their genetic
information stolen and used without permission.
•Many people worry about the safety of modifying food and
medicines using recombinant DNA technology.

16. Safety and Environmental
Issues
 The concern is that the food produced by genetic
engineering could contain toxic proteins or substances that
can cause allergies in people who consume them.
 Genetically engineered crops could spread into the wild
and wipe out native plant species.
 Transgenic crops could transmit their new genes to other
species in neighboring areas.
Example: super-weeds produced by rice and lawn grasses
exchanging pollen with native species
 Foods produced by transgenic crops can be sold without
special permits or labels if the product is identical to
products produced by non-transgenic crops.
Example: corn, tomatoes

17. Advancement to Recombinant DNA
Technology:-Genome editing
 Altering the sequence of DNA “in situ”
 Targeted mutagenesis
 Knock-outs
 Point mutations
 Gene insertions or “trait landing pads”
 Ideally leaving no transgene footprint

18. Genome editing tools
 Meganucleases: 1990s
 Oligonucleotide-directed mutagenesis:
late 1990s
 Zinc finger nucleases (ZFNs): mid 2000s
 Transcription activator like effector
nucleases (TALENs): early 2010s
 Clustered regularly spaced short
palindromic repeats (CRISPR): 2013

19. Way forward
 Technological innovations will continue
 Until disaster strikes
 And then innovations will resume
 But they will be (more) regulated
 So there is a balance between
innovation potential risks and
regulation  ensure safety

20. Trends
 Life science/ Biotechnology synthetic biology
 Designed components
 More precise gene integration and regulation
 Building a crop from scratch?
 Transgenics  genome editing
 Regulations? What about a tiered approach?
 Increasing gap between public’s knowledge of science and technology
and science and technology advances
 But…patents expire and economics shift…times change

21. www.biotecnika.org
Thank you
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