GENETIC ENGINEERING Introduction and Application

GENETIC ENGINEERING
DR. DEEPAK P

Unit 1- Introduction to rDNA Technology and
Genetic Engineering
Historical account of the advent of rDNA technology
(Stanley Cohen and Herbert Boyer Experiment); Basic steps
in gene cloning- Restriction Digestion, Ligation.
Transformation and Screening; Reporter and Marker gene;
Genomic DNA and cDNA libraries- construction and
applications; Ethical implications of biotechnological
products and techniques.

Unit 2- Enzymes in Genetic Engineering
Restriction-Modification System in E.coli; Nucleases- Exonucleases and
Endonucleases, Restriction Enzymes- Type II Structure and Catalysis,
Recognition Sequences, Star activity of Restriction Enzymes; Single-strand
specific nucleases- S1 and Mungbean nuclease; End Modifying Enzymes:
Terminal Transferase, T4 Polynucleotide Kinase, Alkaline Phosphatases;
Methylases: CpGMethylase, Dam Methylase, DcmMethylase; Polymerases:
DNA Pol I, Taq andPfu Polymerases, Klenow Fragment; Reverse
Transcriptases- AMV and MLV; Ligases: T4 and E.coli DNA Ligase, T4 RNA
Ligase; Topoisomerases: Type I and Type II, RNases, Basics of Crispr-Cas9,
ZFNs, TALENs.

Unit 5: Molecular Techniques and Applications
of Genetic engineering
Radioactive and non-radioactive probes; Blotting
Techniques-Southern, Western and northern;
Nucleic Acid Sequencing: Introduction, Sanger’s
method and its automation, Pyro sequencing and
NGS- Illumina Sequencing; Protein Engineering-
Site directed mutagenesis; PCR and its variants;
q-PCR; Genome editing.

Unit 1
rDNA technology, also known as recombinant DNA technology or genetic
engineering, is a revolutionary branch of biotechnology that involves the
manipulation of DNA molecules to create new genetic combinations that do not
occur naturally.
This technology enables scientists to introduce specific genes from one organism
into another, thereby transferring desirable traits and producing organisms with
novel characteristics.

The process of rDNA technology
involves several key steps:

Isolation of DNA: The first step is to extract DNA from the source organism. This
may be done using various methods, such as cell lysis and purification, to obtain
the desired gene of interest.
Cutting DNA: Specific enzymes called restriction enzymes are used to cut DNA at
precise locations. These restriction enzymes recognize specific DNA sequences
and create "sticky ends," which can easily bind to complementary DNA fragments.
Gene of Interest: The gene of interest, containing the desired trait, is isolated and
cut out from the source DNA using the same restriction enzymes. This fragment
contains the information required to produce the desired protein or trait.
DNA Ligase: The isolated gene is then inserted into a vector, which is typically a
small, circular piece of DNA called a plasmid. DNA ligase is used to join the gene
with the vector, creating a recombinant DNA molecule.

Transformation: The recombinant DNA molecule is introduced into
the host organism, often a bacterium or yeast cell. This process is
known as transformation. The host cell takes up the recombinant
DNA and becomes capable of expressing the gene of interest.
Selection: To identify which host cells have successfully incorporated
the recombinant DNA, researchers use selectable markers, such as
antibiotic resistance genes. Only the transformed cells with the
desired gene will survive when exposed to the appropriate antibiotic.
Expression: Once the recombinant DNA has been integrated into the
host organism, it will start expressing the gene of interest, leading to
the production of the desired protein or trait.

Genetic engineering
Genetic engineering is a broad term that encompasses
various techniques and processes used to modify the genetic
material of an organism.
It involves the manipulation of DNA, the hereditary material
of living organisms, to introduce specific traits or characteristics
into an organism or alter its existing genetic makeup.
This field of biotechnology has revolutionized many aspects of
science, agriculture, medicine, and industry.

rDNA technology has numerous applications in various fields, including
agriculture, medicine, and industry.
It has enabled the development of genetically modified crops with enhanced
traits like pest resistance and increased nutritional content.
In medicine, rDNA technology is utilized to produce therapeutic proteins,
vaccines, and gene therapies.
Additionally, it plays a crucial role in the production of enzymes, hormones,
and other industrial products.
However, the use of rDNA technology also raises ethical and safety
considerations, which necessitates rigorous regulations and guidelines to ensure
responsible research and application.

The key techniques and methods
involved in genetic engineering include:

Genetic engineering has far-reaching
applications in various fields

Historical account of the advent of
rDNA technology
Discovery of DNA as the Genetic Material (1869-1953): In 1869, Swiss physician Friedrich
Miescher discovered a substance rich in phosphorus and nitrogen, which he called
"nuclein" and is now known as deoxyribonucleic acid (DNA).
However, it wasn't until the 20th century that scientists began to understand the
significance of DNA as the carrier of genetic information.
In 1953, James Watson and Francis Crick proposed the double helix structure of DNA,
elucidating its role as the hereditary material.

rDNA technology
Isolation of Restriction Enzymes (1960s): In the 1960s, Werner Arber,
Hamilton O. Smith, and Daniel Nathans discovered restriction enzymes,
which are enzymes capable of cutting DNA at specific sequences.
This discovery was a critical step in the development of rDNA
technology, as it allowed scientists to precisely manipulate DNA
sequences.

rDNA technology
Creation of the First Recombinant DNA Molecule (1972): In 1972, Paul Berg and his
colleagues created the first rDNA molecule by combining DNA from two different
sources: the DNA of the simian virus 40 (SV40) and the lambda phage.
They used restriction enzymes to cut the DNA at specific sites and then ligated the
DNA fragments together to form a hybrid DNA molecule.
This ground breaking experiment demonstrated the feasibility of combining genetic
material from different sources.

Ref: https://www.si.edu/spotlight/birth-of-biotech/recombinant-drugs

rDNA technology
Development of Plasmid Vectors (1973): In 1973, Stanley Cohen,
Annie Chang, and Herbert Boyer developed plasmid vectors, which are
small, circular DNA molecules found in bacteria.
Plasmids can replicate independently from the bacterial
chromosome and can carry foreign DNA fragments, making them ideal
vehicles for introducing rDNA molecules into host cells.

Development of Plasmid Vectors
(1973):
Pioneros of Development of Plasmid Vectors: Stanley Cohen, Annie Chan & Herbert Boyer

rDNA technology
Invention of DNA Cloning Techniques (1970s): With the discovery of restriction
enzymes and the development of plasmid vectors, researchers began to develop
DNA cloning techniques. These techniques allowed scientists to insert specific
DNA fragments into plasmids, which were then introduced into bacteria.
The bacteria acted as "biological factories" to produce copies of the inserted
DNA, enabling the amplification and isolation of specific genes.

rDNA technology
First Transgenic Organism (1974): In 1974, Rudolf Jaenisch and Beatrice Mintz
created the first transgenic mouse.
They introduced foreign DNA into mouse embryos, and the transgenic mice that
resulted from these embryos expressed the introduced DNA in their tissues.
This experiment demonstrated the potential for genetically engineering whole
organisms.

First Transgenic Organism (1974):
The genetically modified mouse in which a gene affecting hair growth
has been knocked out (left) shown next to a normal lab mouse

rDNA technology
Introduction of DNA Sequencing Methods (1977): The development of
DNA sequencing techniques, notably the Sanger sequencing method, by
Frederick Sanger and colleagues in 1977, allowed scientists to read and
decipher the precise order of nucleotides in a DNA molecule.
This breakthrough significantly advanced the understanding of DNA
structure and paved the way for the genetic engineering of specific genes.

rDNA technology
Commercialization of rDNA Products (1980s): During the 1980s, the
commercial applications of rDNA technology began to emerge.
Companies started producing genetically engineered
pharmaceuticals, such as insulin and human growth hormone, using
genetically modified bacteria to produce these proteins in large
quantities.

rDNA technology
Expansion of rDNA Applications (1990s and Beyond): The
development of rDNA technology continued to advance, with
applications expanding to include genetically modified crops,
gene therapy for human diseases, the creation of genetically
engineered animals, and various biotechnological innovations.

Stanley Cohen and Herbert
Boyer Experiment
HERBERT BOYER AND STANLEY COHEN

Introduction
 Recombinant DNA (rDNA) technology is
one of the most significant advancements in
molecular biology and biotechnology.
 The development of rDNA technology in the
early 1970s revolutionized genetic research
and has led to numerous applications in
medicine, agriculture, and industry.

1. Early Discoveries Leading to rDNA
Technology
1953: Discovery of the DNA Structure
o
Scientists: James Watson and Francis Crick, with
crucial contributions from Rosalind Franklin and
Maurice Wilkins.
o
Significance: Unveiling the double-helix structure of
DNA laid the foundation for understanding genetic
material and its replication.

1. Early Discoveries Leading to rDNA
Technology
1960s: Understanding Genetic Code and Gene Expression
o
Key Contributions:

Marshall Nirenberg and Har Gobind Khorana:
Deciphered the genetic code.

Jacob and Monod: Elucidated the mechanisms of
gene regulation and operons.
o
Impact: Provided insights into how genes control protein
synthesis, setting the stage for manipulating genetic
material.

Har Gobind Khorana
Marshall Nirenberg

2. Pioneering Experiments in rDNA
Technology

1972: Isolation of Restriction Enzymes
o
Scientists: Hamilton O. Smith, Daniel Nathans, and Werner
Arber.
o
Discovery: Restriction enzymes can cut DNA at specific
sequences, allowing for precise manipulation of DNA.
o
Impact: Enabled the cutting and pasting of DNA fragments, a
critical step in creating recombinant DNA.

2. Pioneering Experiments in rDNA
Technology

1972: Creation of Recombinant DNA Molecules
o
Scientists: Paul Berg and his colleagues at Stanford
University.
o
Experiment: Combined DNA from the monkey virus
SV40 with the lambda virus DNA.
o
Significance: Demonstrated the feasibility of combining
DNA from different sources, creating the first recombinant
DNA molecules.

3. The Cohen-Boyer Experiment and
the Birth of rDNA Technology

1973: First Successful rDNA Experiment
o
Scientists: Stanley N. Cohen and Herbert W. Boyer.
o
Experiment:

Process:
• Plasmid Isolation: Isolated plasmids from bacterial cells.
• Restriction Digestion: Used restriction enzymes to cut the plasmid and foreign
DNA.
• Ligation: Combined the plasmid and foreign DNA fragments using DNA ligase.
• Transformation: Introduced the recombinant plasmid into Escherichia coli (E. coli)
bacteria.
• Screening: Identified bacteria that contained and expressed the recombinant DNA.

Result: The bacteria expressed the foreign gene, demonstrating the successful creation
and functionality of recombinant DNA.

4. Further Developments and Impact
of rDNA Technology

Mid-1970s: Development of rDNA Tools
o
Advances:

Plasmid Vectors: Enhanced methods for cloning and
expressing genes.

Gene Cloning Techniques: Refined processes for inserting
and replicating genes in host organisms.
o
Impact: Improved the efficiency and reliability of rDNA
technology.

4. Further Developments and Impact
of rDNA Technology

1980: Commercialization and Biotechnology
Revolution
o
Milestone: First genetically engineered human
insulin (Humulin) approved for medical use.
o
Impact: Demonstrated the practical applications of
rDNA technology in medicine, leading to the
biotechnology industry's growth.

5. Ethical and Safety Considerations

Concerns:
o
Biohazards: Potential risks of creating harmful organisms or
unintended genetic consequences.
o
Ethical Issues: Moral implications of genetic manipulation, including
GMOs and gene therapy.
o
Regulation: Development of guidelines and oversight for safe and
ethical use of rDNA technology.

Key Responses:
o
Asilomar Conference (1975): Scientists convened to discuss the safe
use of rDNA technology and established safety guidelines.
o
Government Regulations: Implementation of policies to ensure the
responsible use of genetic engineering in research and industry.

6. Conclusion
 The advent of rDNA technology in the early 1970s marked a
transformative period in biological sciences.
 The pioneering experiments by Cohen and Boyer laid the
groundwork for modern genetic engineering, leading to
significant advances in medicine, agriculture, and
biotechnology.
 While the technology has opened numerous opportunities, it
also brought ethical and safety considerations that continue
to shape its development and application.

Restriction Digestion
Restriction digestion, also known as restriction enzyme digestion, is
a crucial molecular biology technique that involves cutting DNA at
specific recognition sequences using restriction enzymes.
These enzymes are naturally occurring proteins found in bacteria
and are used by the bacteria as a defense mechanism against
invading viral DNA.
Restriction enzymes recognize specific short DNA sequences and
cleave the DNA at or near these recognition sites, creating DNA
fragments with "sticky ends" or "blunt ends."

https://www.addgene.org/protocols/restriction-digest/

The basic steps involved in restriction
digestion are as follows:
1. Isolation of DNA: The first step is to isolate the DNA that you wish
to digest. This DNA can come from various sources, such as
genomic DNA, plasmids, or PCR products.
2. Selection of Restriction Enzymes: Choose the appropriate
restriction enzyme(s) based on the specific recognition sequence(s)
present in the DNA you want to cut. Different restriction enzymes
recognize and cut different DNA sequences. The recognition
sequences are typically palindromic, meaning the sequence reads
the same on both strands in opposite directions (e.g., 5'-GAATTC-
3').

3. Incubation with Restriction Enzymes: Mix the isolated DNA with the
selected restriction enzyme(s) in a reaction buffer containing the necessary
cofactors (e.g., Mg2+).
Incubate the mixture at the optimal temperature for the specific enzyme(s).
The enzyme(s) will bind to their respective recognition sites on the DNA and
cleave the DNA at specific positions within or near these sites.
4. Digestion Products: After incubation, the restriction enzyme(s) will have cut
the DNA into fragments. The resulting DNA fragments may have either "sticky
ends" or "blunt ends," depending on the specific restriction enzyme(s) used.

https://www.researchgate.net/post/
What_are_the_optimum_conditions_for_PCR_product_digesti
on_with_fastdigest_restriction_enzymes

◦ Sticky Ends: Some restriction enzymes create staggered cuts, leaving short,
single-stranded overhangs at the ends of the DNA fragments. These
overhangs can easily base-pair with complementary ends of other DNA
fragments cut with the same restriction enzyme, facilitating the ligation of
DNA fragments together in subsequent steps of molecular cloning.
◦ Blunt Ends: Other restriction enzymes cut both DNA strands at the same
position, generating blunt ends with no overhangs. Blunt-ended DNA
fragments can still be ligated, but they tend to have lower ligation efficiency
compared to sticky-ended fragments.

Ligation
Ligation is a crucial step in molecular biology that involves joining
two or more DNA fragments together to create a recombinant DNA
molecule.
This process is essential in many genetic engineering and cloning
experiments, where DNA fragments with specific genes or regulatory
elements need to be combined to create a functional DNA construct.

The basic steps involved in ligation are
as follows:
DNA Fragments: Obtain the DNA fragments that you want to join together.
These fragments may come from various sources, such as restriction digestion
products, PCR-amplified DNA, or synthesized DNA fragments.
Preparation of Fragments: The DNA fragments need to have compatible ends
to allow successful ligation. If the fragments were generated by restriction
digestion, they may have "sticky ends" with short single-stranded overhangs
that can base-pair with complementary ends of other fragments cut with the
same restriction enzyme. Alternatively, if the fragments have blunt ends,
additional steps may be required to create compatible ends.

as follows:
Ligation Reaction: Mix the DNA fragments together with DNA
ligase enzyme and a ligation buffer. DNA ligase is an enzyme that
catalyzes the formation of phosphodiester bonds between adjacent
DNA fragments. The ligation buffer provides the optimal conditions
for the ligation reaction to occur.
Incubation: The ligation reaction is typically incubated at a specific
temperature for a defined period. During this time, the DNA ligase
facilitates the joining of the DNA fragments, resulting in the
formation of a recombinant DNA molecule.

as follows:
Transformation (optional): If the goal of the ligation is to create a
recombinant plasmid, the ligation product can be introduced into host
cells (e.g., bacteria) in a process called transformation. The
transformed cells will take up the recombinant DNA, and those
containing the desired DNA construct can be selected and cultured.
Verification: After ligation and, if applicable, transformation, the
recombinant DNA needs to be verified to ensure that the correct
fragments were ligated together and that the construct has the
desired sequence. This can be done through various methods, such as
DNA sequencing or restriction analysis.

Transformation Methods
https://images.app.goo.gl/wFH79zTCiwhJ3TtW6

https://images.app.goo.gl/oWk54iVCu4SqB6YV8

GENETIC ENGINEERING Introduction and Application

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