Recombinant DNA Technology

(Introduction)

ZOL1503ET-Unit 2 2021-2022
 Introduction
- Recombinant DNA (rDNA) molecules are DNA molecules formed
by laboratory methods of genetic recombination (such as molecular
cloning) that bring together genetic material from multiple sources,
creating sequences that would not otherwise be found in the genome.

- Recombinant DNA is the general name for a piece of DNA that has
been created by combining at least two fragments from two
different sources.

- Recombinant DNA is possible because DNA molecules from all

organisms share the same chemical structure, and differ only in the
nucleotide sequence within that identical overall structure.

- Recombinant DNA molecules are sometimes called chimeric DNA,

because they can be made of material from two different species.
 Introduction
- The DNA sequences used in the construction of recombinant DNA
molecules can originate from any species.

- For example, plant DNA may be joined to bacterial DNA, or human

DNA may be joined with fungal DNA.

- In addition, DNA sequences that do not occur anywhere in nature may

be created by the chemical synthesis of DNA, and incorporated into
recombinant molecules.

- Using recombinant DNA technology and synthetic DNA, literally any

DNA sequence may be created and introduced into any of a very wide
range of living organisms.
 Construction of
recombinant DNA
 DNA Creation – Molecular cloning
- Molecular cloning is the laboratory process used to create
recombinant DNA.
- It is one of two most widely used methods, along with polymerase
chain reaction (PCR), used to direct the replication of any specific
DNA sequence chosen by the experimentalist.

- There are two fundamental differences between the methods.

- One is that molecular cloning involves replication of the DNA within a

living cell, while PCR replicates DNA in the test tube, free of living
cells.

- The other difference is that cloning involves cutting and pasting DNA
sequences, while PCR amplifies by copying an existing sequence.
 DNA Creation – Molecular cloning
- Formation of recombinant DNA requires a cloning vector, a DNA
molecule that replicates within a living cell.

- Vectors are generally derived from plasmids or viruses, and represent

relatively small segments of DNA that contain necessary genetic
signals for replication, as well as additional elements for convenience
in inserting foreign DNA, identifying cells that contain recombinant
DNA, and, where appropriate, expressing the foreign DNA.

- The choice of vector for molecular cloning depends on the choice of

host organism, the size of the DNA to be cloned, and whether and
how the foreign DNA is to be expressed.
- The DNA segments can be combined by using a variety of methods,
such as restriction enzyme/ligase cloning or Gibson assembly.
 DNA Creation – Molecular cloning
- In standard cloning protocols, the cloning of any DNA fragment
essentially involves seven steps:

- (1) Choice of host organism and cloning vector

- (2) Preparation of vector DNA
- (3) Preparation of DNA to be cloned
- (4) Creation of recombinant DNA
- (5) Introduction of recombinant DNA into the host organism
- (6) Selection of organisms containing recombinant DNA
- (7) Screening for clones with desired DNA inserts and biological
properties.
 DNA Creation – Molecular cloning
- In standard cloning protocols, the cloning of any DNA fragment
essentially involves seven steps:

- (1) Choice of host organism and cloning vector

- (2) Preparation of vector DNA
- (3) Preparation of DNA to be cloned
- (4) Creation of recombinant DNA
- (5) Introduction of recombinant DNA into the host organism
- (6) Selection of organisms containing recombinant DNA
- (7) Screening for clones with desired DNA inserts and biological
properties.
 Molecular Cloning
- The use of the word cloning refers to the fact that the method
involves the replication of one molecule to produce a population of
cells with identical DNA molecules.
 Molecular Cloning
- Although the detailed planning of the cloning can be done in any text
editor, together with online utilities for e.g. PCR primer design,
dedicated software exist for the purpose.

- Software for the purpose include for example ApE (open source),
DNAStrider (open source), Serial Cloner (gratis) and Collagene (open
source).

- Proteins that can result from the expression of recombinant DNA

within living cells are termed recombinant proteins.

- When recombinant DNA encoding a protein is introduced into a host

organism, the recombinant protein is not necessarily produced.
- Expression of foreign proteins requires the use of specialized
expression vectors and often necessitates significant restructuring by
foreign coding sequences.
 Choice of Organisms and Vectors
- Although a very large number of host organisms and molecular
cloning vectors are in use, the great majority of molecular cloning
experiments begin with a laboratory strain of the bacterium E.
coli (Escherichia coli) and a plasmid cloning vector.

- E. coli and plasmid vectors are in common use because they are
technically sophisticated, versatile, widely available, and offer rapid
growth of recombinant organisms with minimal equipment.
- If the DNA to be cloned is exceptionally large (hundreds of thousands
to millions of base pairs), then a bacterial artificial chromosome
(BAC) or yeast artificial chromosome (YAC) vector is often chosen.
 Specialized host-vector systems
- Specialized applications may call for specialized host-vector systems.

- For example, if the experimentalists wish to harvest a particular

protein from the recombinant organism, then an expression vector
is chosen that contains appropriate signals for transcription and
translation in the desired host organism.

- Alternatively, if replication of the DNA in different species is desired

(for example, transfer of DNA from bacteria to plants), then a
multiple host range vector (also termed shuttle vector) may be
selected.

- In practice, however, specialized molecular cloning experiments

usually begin with cloning into a bacterial plasmid, followed by
subcloning into a specialized vector.
 Transformation
- The first step in transformation is to select a piece of DNA to be
inserted into a vector.

- The second step is to cut that piece of DNA with a restriction enzyme
and then ligate the DNA insert into the vector with DNA Ligase.

- The insert contains a selectable marker which allows for

identification of recombinant molecules.

- An antibiotic marker is often used so a host cell without a vector dies

when exposed to a certain antibiotic, and the host with the vector
will live because it is resistant.

- The vector is inserted into a host cell, in a process called

transformation.
 Transformation
- One example of a possible host cell is E. Coli.

- The host cells must be specially prepared to take up the foreign DNA.

- Selectable markers can be for antibiotic resistance, color changes, or

any other characteristic which can distinguish transformed hosts from
untransformed hosts.

- Different vectors have different properties to make them suitable to

different applications.

- Some properties can include symmetrical cloning sites, size, and high
copy number.
 Non-bacterial Transformation
- This is a process very similar to Transformation.

- The only difference between the two is non-bacterial does not use
bacteria such as E. Coli for the host.

- In microinjection, the DNA is injected directly into the nucleus of the

cell being transformed.

- In biolistics, the host cells are bombarded with high velocity

microprojectiles, such as particles of gold or tungsten that have been
coated with DNA.
DNA Microinjection
Biolistics (Gene Gun)
- A gene gun is used for delivery
of exogenous DNA to cells.
This method is known as
'biolistics’.

- Gene guns can be used

effectively on most cells but
are mainly used on plant cells.

- Step 1: The gene gun

apparatus is ready to fire.

- Step 2: Helium fills the

chamber and pressure builds
against the rupture disk.
Biolistics (Gene Gun)
- Step 3: The pressure
eventually reaches the point
where the rupture disk breaks,
and the resulting burst of
helium propels the DNA/gold-
coated macrocarrier ('Plastic
Disk') into the stopping screen.

- Step 4: When the macrocarrier

hits the stopping screen, the
DNA-coated gold particles are
propelled through the screen
and into the target cells.
 Phage Introduction
- Phage introduction is the process of transfection, which is equivalent
to transformation, except a phage is used instead of bacteria.

- In vitro packaging of a vector is used.

- This uses lambda or MI3 phages to produce phage plaques which

contain recombinants.

- The recombinants that are created can be identified by differences in

the recombinants and non-recombinants using various selection
methods.
 Importance of Recombinant DNA Technology
- Recombinant DNA has been gaining in importance over the last few
years, and recombinant DNA will only become more important in the
21st century as genetic diseases become more prevalent and
agricultural area is reduced.

- Below are some of the areas where Recombinant DNA will have an
impact.
Better Crops (drought & heat resistance)
Recombinant Vaccines (Hepatitis B)
Prevention and cure of sickle cell anemia
Prevention and cure of cystic fibrosis
Production of clotting factors
Production of insulin
Production of recombinant pharmaceuticals
Plants that produce their own insecticides
Germ line and somatic gene therapy
 Applications of Recombinant DNA Technology
- Recombinant DNA is widely used in biotechnology, medicine and
research.

- Today, recombinant proteins and other products that result from the
use of DNA technology are found in essentially every western
pharmacy, physician or veterinarian office, medical testing laboratory,
and biological research laboratory.

- In addition, organisms that have been manipulated using

recombinant DNA technology, as well as products derived from those
organisms, have found their way into many farms, supermarkets,
home medicine cabinets and and even pet shops, such as those that
sell GloFish and other genetically modified animals.
 Applications of Recombinant DNA Technology
- The most common application of recombinant DNA is in basic
research, in which the technology is important to most current work
in the biological and biomedical sciences.
- Recombinant DNA is used to identify, map and sequence genes, and
to determine their function.

- rDNA probes are employed in analyzing gene expression within

individual cells, and throughout the tissues of whole organisms.

- Recombinant proteins are widely used as reagents in laboratory

experiments and to generate antibody probes for examining protein
synthesis within cells and organisms.
 Applications – Recombinant Chymosin
- Found in rennet, chymosin is an enzyme required to manufacture
cheese.

- Rennet is a complex set of enzymes produced in the stomachs of

ruminant mammals.

- Chymosin, its key component, is a protease enzyme that curdles the

casein in milk.

- It was the first genetically engineered food additive used

commercially.

- Traditionally, processors obtained chymosin from rennet, a

preparation derived from the fourth stomach of milk-fed calves.
 Applications – Recombinant Chymosin
- Scientists engineered a non-pathogenic strain (K-12) of E. coli bacteria
for large-scale laboratory production of the enzyme.

- This microbiologically produced recombinant enzyme, identical

structurally to the calf derived enzyme, costs less and is produced in
abundant quantities.

- Today about 60% of U.S. hard cheese is made with genetically

engineered chymosin.

- In 1990, FDA granted chymosin “generally recognized as safe” (GRAS)

status based on data showing that the enzyme was safe.
Applications – Recombinant Chymosin
 Applications – Recombinant Human Insulin
- Almost completely replaced insulin obtained from animal sources
(e.g. pigs and cattle) for the treatment of insulin-dependent diabetes.

- A variety of different recombinant insulin preparations are in

widespread use.

- Recombinant insulin is synthesized by inserting the human insulin

gene into E. coli, or yeast (Saccharomyces cerevisiae) which then
produces insulin for human use.
 Applications –
Recombinant Human
Insulin
 Applications – Recombinant Human Growth Hormone

- Administered to patients whose pituitary glands generate

insufficient quantities to support normal growth and development.

- Before recombinant human growth factor (HGH) became available,

HGH for therapeutic use was obtained from pituitary glands of
cadavers.

- This unsafe practice led to some patients developing Creutzfeldt-

Jakob.

- Recombinant HGH eliminated this problem, and is now used

therapeutically.
- It has also been misused as a performance-enhancing drug by
athletes and others.
 Applications – Recombinant Blood Clotting Factor VIII

- A blood-clotting protein that is administered to patients with forms of

the bleeding disorder hemophilia, who are unable to produce factor
VIII in quantities sufficient to support normal blood coagulation.
- Before the development of recombinant factor VIII, the protein was
obtained by processing large quantities of human blood from
multiple donors, which carried a very high risk of transmission of
blood borne infectious diseases, for example HIV and hepatitis B.
 Applications – Recombinant Hepatitis B Vaccine

- Hepatitis B infection is controlled through the use of a recombinant

hepatitis B vaccine, which contains a form of the hepatitis B virus
surface antigen that is produced in yeast cells.

- The development of the recombinant subunit vaccine was an

important and necessary development because hepatitis B virus,
unlike other common viruses such as olio virus, cannot be grown in
vitro.
 Applications – Diagnosis of infection with HIV

- Each of the three widely used methods for diagnosing HIV infection
has been developed using recombinant DNA.

- The antibody test (ELISA or western blot) uses a recombinant HIV

protein to test for the presence of antibodies that the body has
produced in response to an HIV infection.

- The DNA test looks for the presence of HIV genetic material using
reverse transcription polymerase chain reaction (RT-PCR).

- Development of the RT-PCR test was made possible by the

molecular cloning and sequence analysis of HIV genomes.
 Applications – Golden Rice
- A recombinant variety of rice that has been engineered to express
the enzymes responsible for beta-carotene biosynthesis.
- This variety of rice holds substantial promise for reducing the
incidence of vitamin A deficiency in the world's population.
- In 2018, Canada and the United States approved golden rice for
cultivation, with Health Canada and the US Food and Drug
Administration declaring it safe for consumption.

- In 2019, it was approved for direct use as human food and animal
feed or for processing in the Philippines.
 Applications – Herbicide Resistant Crops
- Commercial varieties of important agricultural crops (including soy,
maize/corn, sorghum, canola, alfalfa and cotton) have been
developed that incorporate a recombinant gene that results in
resistance to the herbicide glyphosate (trade name Roundup), and
simplifies weed control by glyphosate application.

- These crops are in common commercial use in several countries.

Roundup ready
 Applications – Insect Resistant Crops
- Bacillus thuringiensis is a bacterium that naturally produces a protein
(Bt toxin) with insecticidal properties.
- The bacterium has been applied to crops as an insect-control strategy
for many years, and this practice has been widely adopted in
agriculture and gardening.

- Recently, plants have been developed that express a recombinant form

of the bacterial protein, which may effectively control some insect
predators.

- Environmental issues associated with the use of these transgenic crops

have not been fully resolved.
Applications – Insect Resistant Crops

Bt toxins present in peanut leaves

(bottom dish) protect it from extensive
damage caused to unprotected peanut
leaves by lesser cornstalk borer larvae
 Indian Controversies
- In India, GM cotton yields in Maharashtra, Karnataka and Tamil Nadu
resulted in an average 42% increase in yield in 2002, the first year of
commercial planting.

- A severe drought in Andhra Pradesh that year prevented any increase

in yield, because the GM strain was not drought tolerant. Drought-
tolerant variants were later developed.

- Driven by substantially reduced losses to insect predation, by 2011

88% of Indian cotton was modified.

- A study from 2002 through 2008 on the economic impacts of Bt

cotton in India, showed that Bt cotton increased yields, profits and
living standards of smallholder farmers.
 Indian Controversies
- However, recently cotton bollworm has been developing resistance to
Bt cotton.

- Consequently, in 2012 Maharashtra banned Bt cotton and ordered

an independent socioeconomic study of its use.

- Indian regulators cleared the Bt brinjal, a genetically modified

eggplant, for commercialisation in October 2009.

- After opposition by some scientists, farmers and environmental

groups, a moratorium was imposed on its release in February 2010
"for as long as it is needed to establish public trust and confidence".
 Indian Controversies

- As of 1 January 2013, all foods containing GMOs must be labelled.

- The Legal Metrology (Packaged Commodities) Rules, 2011 states that

"every package containing the genetically modified food shall bear at
the top of its principal display panel the letters 'GM.’

- On March 21, 2014, the Indian government revalidated 10 GM-based

food crops and allowed field trials of GM food crops, including wheat,
rice and maize.
 Applications – Production of Vaccines
- Vaccines are the chemical preparations containing a pathogen in
attenuated (or weakened) or inactive state that may be given to
human beings or animals to confer immunity to infection.

- A number of vaccines have been synthesized biologically through

recDNA technology, these vaccines are effective against numerous
serious diseases caused by bacteria, viruses or protozoa.

- These include vaccines for polio, malaria, cholera, hepatitis, rabies,

smallpox, etc.

- The generation of DNA vaccines has revolutionized the approach of

treatment of infectious diseases.

- DNA-vaccine is the preparation that contains a gene encoding an

immunogenic protein from the concerned pathogen.
 Applications – Production of Antibiotics
- Antibiotics produced by microorganisms are very effective against
different viral, bacterial or protozoan diseases.

- Some important antibiotics are tetracyclin, penicillin, streptomycin,

novobiocin, bacitracin, etc.

- recDNA technology helps in increasing the production of antibiotics

by improving the microbial strains through modification of genetic
characteristics.
 Applications – Production of Antibiotics
- Antibiotics produced by microorganisms are very effective against
different viral, bacterial or protozoan diseases.

- Some important antibiotics are tetracyclin, penicillin, streptomycin,

novobiocin, bacitracin, etc.

- recDNA technology helps in increasing the production of antibiotics

by improving the microbial strains through modification of genetic
characteristics.
END