DNA TECHNOLOGY Micropipette - used to accurately and precisely transfer

volumes of liquid in the microliter range.

- use of DNA to improve quality of life
1. Gel Electrophoresis
the study and manipulation of genetic material
-device used for profiling DNA fragments based on size and
- use of DNA to answer unknown questions about life electric charge in an electric field.
specially for the treatment of disease
2. Restriction enzymes (molecular scissors)
2 types of DNA technology
- an enzyme isolated from bacteria that cuts
With recombinant DNA
- DNA molecules at specific sequences
- creates DNA hybrid or altered genes
3. DNA Ligase (genetic paste)
- DNA are isolated from two different cells and joined
together that do not normally occur - Specific type of enzyme that facilitates the joining if DNA
strands together
Without recombinant
4. Host
- Do not warrant the use of creation of recombinant DNA
- enables the multiplication of molecular sequence
- genes may only be isolated, identified, sequenced and
amplified - Prokaryotic host or Eukaryotic host

TYPES OF DNA TECHNOLOGY 5. Plasmid

* With recombinant DNA - commonly known as vectors

modify - Genetic engineering - serves as tool to clone, transter, and manipulate genes

fix - Gene and radiation therapy 6. Polymerase Chain Reaction (PCR)

medicine - Pharmacogenomics - a laboratory technique used to amplify DNA sequences

unnatural - Making of GMOs

* Without recombinant Genetic Engineering

characteristics - DNA screening - also called genetic modification or genetic manipulation

identity - DNA fingerprinting - direct organism's biotechnology

twin - Gene/human/cell cloning manipulation genes an using process of using recombinant

natural - Selective breeding - DNA (rDNA) technology to alter the genetic makeup of an
organism
COMMONLY USED TOOLS IN GENETIC ENGINEERING

Autoclave - The main purpose of this device is to sterilize

materials and media under pressure and steam. GMO vs Transgenic Organism

Centrifuge - This device is mainly used in cell culture, nucleic * GMO

acid isolation and in microbiology to separate two liquids in
emulsion form or suspended solids in liquids by the help of GMO is an organism that possesses a genetically modified
the centrifugal force. genome.

UV light - Used to view the separated fragments of DNA in * Transgenic Organism

gel electrophoresis set up.
Transgenic organism is a CMO, but carries DNA sequences or
genes received from a different organism.
Foreign DNA Process of Cloning a Gene in a Bacterial Plasmid

* GMO 1. Isolation of vector and gene-source DNA. - gene of interest

May or may not have foreign DNA 2. Insertion of DNA into the vector. - hybrid DNA

* Transgenic Organism 3. Introduction of the cloning vector into cells. -deliberation

of hybrid DNA
Have foreign DNA
4. Cloning of cells (and foreign genes). - amplification of
genes
Transgenic Organism 5. Identifying cell clones with the right gene. - field testing
- organisms that have altered genomes

- most transgenic organisms are generated in the laboratory Genetically Modified Organisms
for research purposes
British company Oxitec has created genetically modified
- genes of one species can be modified, or genes can be male mosquitoes that carry a "self limiting gene". When they
transplanted from one are released into the wild and mate with females their
offspring do not reach adulthood, so crucially do not
contribute to the spread of the Zika virus. Other researchers
Tracy the Sheep, 1997 are looking at using genetic modification to curb the spread
of malaria.
First transgenic animal to produce a recombinant protein
drug in her milk alpha Engineered to grow at twice the rate of regular salmon, it is
also believed to be the first example of a genetically
-1-antitrypsin (AAT) treatment for emphysema and cystic engineered animal bred and sold for human consumption.
fibrosis.

FLAVR SAVR tomato (1987)

Goats that Produce Important Proteins in their Milk
The FLAVR SAVR™ tomato was developed through the use of
- Goats modified to produce FDA-approved human antisense RNA to regulate the expression of the enzyme
antithrombin (ATryn), which is used to treat a rare blood polygalacturonase (PG) in ripening tomato fruit.
clotting disorder in humans. Goats have also been
genetically modified to produce spider silk, one of the Arctic Apple
strongest materials known to man, in their milk. Proposed
Browning is caused by the release of polyphenol oxidase
uses for this recombinant spider silk range from artificial
(PPO). The gene for PPO release is activated during times of
tendons to bulletproof vest.
stress (slicing, biting, throwing / By identifying the gene that
Golden Rice is responsible for PPO release, biotechnology is used to
silence that gene.
Modified rice that produces beta-carotene, the precursor to
vitamin A. Vitamin A deficiency is a public health problem for
millions of people around the world, particularly in Africa
Steps in DNA Manipulation
and Southeast Asia. Golden rice is still waiting regulatory
approval. In order to produce recombinant DNA, following are
requires:
BT Corn
* Gene of interest, which is to be cloned
These GE plants produce crystal (Cry) proteins or toxins
derived from the soil bacterium, Bacillus thuringiensis (Bt), * Molecular scissors, to cut out the gene of interest
hence the common name "Bt maize".
* Molecular carrier (vector), on which gene of interest could
be placed
* An expression system, on which the gene of interest along is now open. The process of joining these two pieces
with the vector is then introduced as a result of which a together using the enzyme DNA ligase is ligation. The
specific product is made resulting DNA molecule is a hybrid of two DNA molecules -
the interest molecule and the vector. In the terminology of
How to get a genes: genetics this intermixing of different DNA strands is called
recombination.
In the process of recombinant DNA technology, first of all
we'll have to get a gene which is to be cloned. There are two Hence, this new hybrid DNA molecule is also called a
ways to get the gene of interest. recombinant DNA molecule and the technology is referred
to as the recombinant DNA technology.
* To Isolate It from The Chromosome: Genes can be isolated
from the chromosomes by cutting the chromosomes on the 5. Insertion of Recombinant DNA into Host - In this step, the
flanking sites of the gene using special enzymes known as recombinant DNA is introduced into a recipient host cell
restriction endonucleases. mostly, a bacterial cell. This process is called transformation.
Bacterial cells do not accept foreign DNA easily. Therefore,
* Chemical Gene Synthesis by Messenger RNA (mRNA): If
they are treated to make them competent to accept new
the genes are small or for some reason we can't isolate from
DNA. The processes used may be thermal shock, Cat+ ion
chromosomes, they can also be synthesized chemically in
treatment, and electroporation.
the laboratory. To make a gene chemically in laboratory,
messenger RNA (mRNA) is used. We'll use the reverse Ways of Hybrid DNA deliberation
transcriptase enzyme method. Reverse transcriptase is the
enzyme which forms DNA from an RNA template in reverse * Bacteria - transformation- exposure to heat and cold to
transcription. This DNA molecule is called complementary open up and close the bacterial pores
DNA (cDNA) and is mainly associated with retro viruses.
* Plants - exposure to radiation (particle bombardment
device) exposure to Agrobacterium tumefaciens

Steps of Genetic Recombination Technology * Animals - microinjection

1. Isolation of Genetic Material - involves enzymes such as 6. Isolation of Recombinant Cells-The transformation
restriction enzymes. process generates a mixed population of transformed and
non-transformed host cells. The selection process involves
2. Restriction Enzymes Digestion - technique 'Agarose Gel filtering the transformed host cells only. For isolation of
Electrophoresis' reveals the progress of the restriction recombinant cells from non-recombinant cells, a marker
enzyme digestion - it allows separating and cutting out the gene of the plasmid vector is employed.
digested DNA fragments. The vector DNA is also processed
using the same procedure. 7. Obtaining or culturing the Foreign Gene product - When
you insert a piece of alien DNA into a cloning vector and
3. Amplification Using PCR transfer it into a bacterial cell, the alien DNA gets multiplied.
The ultimate aim is to produce a desirable protein
PCR reactions are run on thermal cyclers using the following
expression.
components:
The cells harboring cloned genes of interest are grown on a
•Template - DNA to be amplified
small scale in the laboratory. These cell cultures are used for
• Primers - small, chemically synthesized oligonucleotides extracting the desired protein using various separation
that complementary to a region of the DNA. techniques.

•Enzyme - DNA Polymerase

•Nucleolides - needed to extend the primers by the enzyme.

The cut fragments of DNA can be amplified using PCR and

then ligated with the cut vector.

4. Ligation of DNA Molecules - The purified DNA and the

vector of interest are cut with the same restriction enzyme.
This gives us the cut fragment of DNA and the cut vector that