RECOMBINANT

DNA
Learning Objectives

At the end of the presentation, you should be able to:

 outline the processes involved in genetic engineering

 discuss the application of recombinant DNA
 Recombinant DNA are molecules of DNA from two different species that
are inserted into a host organism to produce new genetic combinations
that are of value to science, medicine, agriculture, and industry. Since the
focus of all genetics is the gene, the fundamental goal of laboratory
geneticists is to isolate, characterize, and manipulate genes.
Recombinant DNA is also known as Genetic Engineering, Genetic
Modification and Biotechnology. It is a set of techniques that are used to
achieve one or more of three goals:

A. To reveal the processes of how genes are inherited and expressed;

B. To provide effective treatment for various diseases (particularly genetic
disorders);
C. To generate economic benefits which include improved plants and
animals for agriculture, and efficient production of valuable
biopharmaceuticals.
PROCESSES/ETEPS INVOLVED IN GENETIC ENGINEERING

The process of genetic engineering is not a simple task, it requires complex

machinery and innovative minds. Since farming began, humans have been
selectively breeding different plants, doing so provided more food and
better food for all. This process is genetic engineering in much simpler
terms than we think of it today.

Now, when one thinks of genetic engineering, they are more likely to
picture a complicated science which involves altering the very building
blocks of life. The are several steps in the process of genetic engineering.
Scientist follow a step-by-step process in order to alter the DNA of an
organism.
This diagram/illustration seen on the previous page demonstrated
how the plasmid of an E.coli bacterium was isolated same is true
with the DNA of the cell containing gene of interest. When already
isolated, the plasmid is cut with enzyme 9 and the cell’s DNA with
enzyme is also cut. Then, the cut targeted fragments are combined
(plasmid DNA). Once combined together, a DNA ligase is added
which closes the circle with covalent bonds. Thus, the gene of
interest is already one with the plasmid forming a recombinant
DNA. Where this can also be used or done in Gene Cloning as
shown in the illustration below.
 Recombinant DNA indeed has no ordinary process.
In order to achieve something worthwhile, the
process should be well done and followed. If not,
then the experiment is considered failed, and
scientist will have to do the process repeatedly until
the desired product is achieved.
 3 MAIN TOOLS
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.
LIGASES:
DNA ligases play an essential role in maintaining genomic integrity by
joining breaks in the phosphodiester backbone of DNA that occur during
replication and recombination, and as a consequence of DNA damage and its
repair.
RESTRICTION ENZYMES:
A restriction enzyme is a protein isolated from bacteria that cleaves DNA
sequences at sequence-specific sites, producing DNA fragments with a known
sequence at each end. The use of restriction enzymes is critical to certain
laboratory methods, including recombinant DNA technology and genetic
engineering.
The five key tools involved in recombinant DNA
technology are as follows:

1. Enzymes involved in DNA manipulation

2. Cloning vectors
3. Gel electrophoresis
4. PCR
5. Host organism
Enzymes involved in DNA manipulation:
Enzymes involved in DNA manipulation are as follows:

1. Restriction enzymes
 These enzymes serve as molecular scissors to cut
DNA into defined fragments.
 These enzymes recognize special palindromic
nucleotide sequences of 4-8 nucleotides in length in
DNA.
 Restriction endomucelases (a type of restriction
enzyme) are used to isolate gene of interest and to
create sticky ends in plasmid vectors.
 Example - Eco R I
2. Polymerase enzyme

 These enzymes cause extension of DNA

strands in the 5’ to 3’ direction by using
template DNA.
 E.g., Taq polymerase in PCR
3. Ligase enzyme

 The DNA ligase joins two DNA strands by

forming a phosphodiester bond between
them.
 It is known as molecular glue.
 It is used to join genes of interest with the
cloning vector.
 E.g., T4 DNA ligase is used in rDNA
technology.
Cloning vector :
Cloning vehicles that carry the gene of interest into the recipient cell are
called cloning vectors.

Different types of cloning vectors are listed below:

 Plasmids and viruses are commonly used as vectors.
 Plasmids are extra chromosomal circular DNA present in prokaryotic
organisms such as bacteria, that are capable of replicating themselves.
 The most commonly used vectors are plasmids such as pBR322 and
pUC18.
 Ti plasmid obtained from Agrobacterium is the commonly used
vector for plants.
 Disarmed retroviral vectors are commonly used for animals.
Gel electrophoresis:
 Gel electrophoresis is a technique used to separate DNA fragments
based on their sizes under the influence of an electric field.

PCR:
 Polymerase Chain Reaction is used to amplify the gene of interest.
 Using PCR even small samples of DNA can be amplified and used in
rDNA technology.
 It involves three steps - denaturation, annealing and extension.
Host organism
 The organism in which the gene of interest (with or
without vector) is inserted and multiplied is known as the
host organism.

 Escherichia coli is commonly used for multiplication of

various genes.
 Since the presence of genetic engineering or
recombinant DNA, many Genetically Modified
Organisms (GMO) surfaced. Before that let us first
know what Genetically Modified Organisms are.
 APPLICATIONS OF GENETIC
ENGINEERING
Genetically Modified Organisms (GMOs) are actually
products of the applications or uses of genetic
engineering.
I. Medicine
I.1. Treatment

A. Insulin production Human insulin produced

through genetic engineering since 1982 Human
insulin gene inserted into the bacterium E.coli to
produce synthetic “human” insulin for the treatment
of insulin-dependent diabetes. In the past, insulin
was obtained from a cow or pig pancreas, that has
many problems.
ANOTHER FOR TREATMENT:
B. Producing human growth hormone:
- to treat growth retardation (dwarfism)

C. Producing Follistim Injection:

- (contains the FSH hormone) for treating infertility

D. Making human albumin, anti-hemophilic factors and many

other drugs.

E. Other biopharmaceuticals under development through genetic

engineering, include:

Anti-cancer drug and possible vaccine for AIDS, malaria, COVID19, etc.
I.2. Vaccination
- Today, the microorganism (such as yeast) is used to produce virus
antigen used as vaccine that stimulate the human immune system
against the virus.

- This procedure has been done successfully for the development of

a vaccine against hepatitis B virus (HBV) that is now widely used.

- Genetically engineered vaccines hold great promise for the future.

- GE vaccines may also be useful to prevent diseases that have

resistant to traditional vaccination, including HIV, tuberculosis, etc.
I.3. Gene Therapy
1. Somatic Gene Therapy – is designed to introduce
functional genes to the body which enable the body to
perform normal functions thus, providing correction for
genetic abnormalities. This will cause treating individuals
by targeting the therapy to body cells such as bone marrow
or blood cells.
I.4. TISSUE ENGINEERING
- According to their source, Stem cells are divided into “adult” which are
multipotent, and “embryonic” stem cells, that are mostly pluripotent

- Stem cells can become almost any other cell, they are waiting for a signal that
will tell them what kind of tissue cell to become.

- Stem cells may be useful for the repair of damaged tissues, or may be used to
grow new organs.

- Stem cells in the pulp of primary teeth, characterized as multipotent cells,

have the potential to be used in both dental and medical applications such as:
treatment of periodontal disease, diabetes, spinal cord injury, stroke, heart
attack, burn, rheumatoid arthritis and Parkinson’s and Alzheimer’s diseases,
and regenerate many types of tissue in the body.
II. Agriculture
- Using the genetic engineering techniques widely today, to produce
genetically modified crops/plants.

- In most cases the aim is to introduce a new trait to the plant which does
not occur naturally in the species.

- AIMS of GM crops include;

• Resistance to certain pests, diseases or environmental
conditions.
• Reduction of spoilage.
• Resistance to chemical treatments (e.g. resistance to herbicide)
• Improving the nutrient profile of the crop
- The first GM crop is Flavr Savr
tomato was a tomato engineered to
have a longer shelf life, in 1992.

- In 1995, Bt potato was approved

safe by the Environmental Protection
Agency, with (Bt) protein, wich is
resistant to pest.

- Bt Cotton is a genetically modified

cotton which is resistant to pests

- Golden rice genetically modified

contain beta-carotene (a source of
Vit. A)
Not all what you think looks amazing, yummy and tasty are healthy in this
case. All natural things are still the most amazing things on earth.
However, when it comes to some other applications of Genetic
Engineering, we can say that it is far from amazing.

Moreover, we still have to consider the One who created all these amazing
natural things in the world.

We are done with Recombinant DNA or Genetic Engineering or what we

call Biotechnology. As a student, you should know netter how to take care
of your environment, and be wiser with all the things the you take in, see
to it that they are not that harmful to your body.
THANKIE!!!
Activity 2. Jumbled Words.
Direction: Unscramble the letters to reveal the correct word. Write your answers on the space
provided for.

1. GOLYCHONTOBIE 8. RUVSIES
2. GLONCIN 9. TEJICNION
3. EGEN AREHTYP 10.THOS
4. INERTNIOS 11.NETIRPO
5. TECVRO 12.GANORISMOCRIM
6. MASILPD 13.LINSUNI
7. RETMIUCAB 14.METERNATN
15.NATCIONCAVI