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Lecture 2 (Food Biotechnology).Students

This document provides an overview of genetic engineering techniques, specifically focusing on gene cloning processes, gene transfer methods, and the selection and analysis of clones. It details the steps involved in cloning, including DNA isolation, cutting, and transformation, as well as various methods for introducing foreign genes into cells. Additionally, it discusses the importance of screening and characterization techniques to identify successful recombinant DNA.

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15 views

Lecture 2 (Food Biotechnology).Students

This document provides an overview of genetic engineering techniques, specifically focusing on gene cloning processes, gene transfer methods, and the selection and analysis of clones. It details the steps involved in cloning, including DNA isolation, cutting, and transformation, as well as various methods for introducing foreign genes into cells. Additionally, it discusses the importance of screening and characterization techniques to identify successful recombinant DNA.

Uploaded by

gumiho orange
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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GENETIC ENGINEERING

TECHNIQUE : AN OVERVIEW IN
GENE CLONING
OBJECTIVES
 To describe the processes that are
used in gene cloning
 To describe the processes of
transferring the cloned gene into the
desired organisms
 To describe the methods for selection,
screening and analysis or
characterization of clones
The Central Dogma of Molecular
Biology

Animation
GENE TRANSFER
 The transfer of gene from one organism to
another – 2 processes
 Gene transfer = Transformation
Cloning the gene

Transferring the cloned gene into the


desired organism
Cloning
Isolation of DNA from organism with the desired
gene

Cut the DNA into fragments

Insert fragments into vector

Transform cells with vector

Select or detect or identify cell with desired gene

Culture cells with desired gene


(Amplification)
The General Steps in Gene Cloning
A piece of foreign DNA is isolated and
prepared by restriction digestion for
insertion into an appropriate cloning
vector.

The foreign DNA is assembled into the


cloning vector.

The newly formed DNA is mobilized into


bacteria to reproduce more copies.

The transformed bacteria are grown under


conditions designed to select for the
cells containing the DNA.

The DNA can be re-isolated from the


bacteria and used for a variety of
purposes.
Gene Cloning Experiment

Preparation of purified DNA samples

Construction of the recombinant molecule

Vector DNA to be cloned

Cut at the specific joints (Cutting)

Joined together (joining)

Transformation
Basic Step in Isolation Protocol

Disruption of the starting material

Deproteinisation stage

Precipitation stage

Purification stage
Cutting : Restriction Enzyme & Joining (ligation) : Ligase Enzyme
Restriction enzymes are sequence specific DNA cleavage enzymes that recognize
discrete arrangements of nucleotides (A,C, G, T) along the DNA strand. At each
recognition site (e.g., GGATCC ) the DNA will be cut. The ends of the double-stranded
DNA molecule can be either even (blunt) or uneven (sticky).
TYPES OF TRANSFORMATION
- depends on type of cells
Bacterial cells - Phage - transfection
transformation

Living cells

Non-bacterial cells Yeast


Fungal cells cells

Eukaryote cells (animal Plant cells -


cells) - transfection transformation
METHODS OF BACTERIAL
TRANSFORMATION

Eletroporation – physical
treatment

Chemical
transformation @ Methods
treatment

Gene gun – physical


treatment
TRANSFORMATION OF OTHER
ORGANISMS

Yeast – protoplast,
Animal cells -
lipofection
lipofection

Eukaryotic cells -
electroporation
Other organisms

Fungal -
lipofection
Animals – microprojectiles,
microinjection
High plants- protoplast,
electroporation, plant virus @
bacterial plant pathogen, biolistic
gun
How do you introduce foreign gene
inside the cells?
Techniques to introduce foreign
gene inside the cells.

1. Electroporation
2. Biolistic gun
3. Microinjection
4. Chemical transformation
Electroporation
• The use of an electric field pulse to induce microscopic
pores within a biological membrane and insert the gene
of interest in the cells.
Biolistic Helium-Driven Particle Gun

Biolistics involves the direct delivery of DNA coated metal particles into the cell.
Gold or tungsten particles a few microns in size are coated with recombinant DNA or
RNA and the particles are literally shot into the cell. Once inside the cell, the DNA soaks
off the particle and becomes integrated into the cell genome.

Fires DNA into target cells


For transforming cell that are difficult to
transform e.g plant cells, whole animals

BioRad Particle Gun (left) and Helios Gene Gun (bottom right)
Microinjection

Introduces the transgene DNA at the earliest possible stage


of development of the zygote (fertilized egg)
CHEMICAL TRANSFORMATION @ TREATMENT
Calcium phosphate / calcium chloride
- To increase the ability of foreign gene to be
inserted into a bacterial cell.
LIPOFECTION
DNA mixed with lipid to form liposomes, small vesicles with some
of the DNA inside.DNA-bearing liposomes fuse with cell membrane
carrying DNA inside the cell.
PROTOPLAST TRANSFORMATION (PLANT)

Protoplast = is a cell that its cell wall is removed.


Take up DNA = protoplast + DNA recombinant
Agrobacterium-Mediated Cell Transformation
There are at least three species of Agrobacterium: A. tumefaciens and A.
rhizogenes.
A. tumefaciens initiates gall formation, generally at around the soil surface near
the crown. Hence the name 'crown gall' for the tumor‑like structure that forms
following infection. A. rhizogenes initiates root proliferation.

Agrobacterium contains a large, circular, double‑stranded DNA molecule that can


replicate independently of the A. tumefaciens genome – the Ti plasmid.
When the bacteria infect a plant cell, a segment of the Ti plasmid called the T-DNA
excises and incorporates into the host cell genome. This aspect of Ti plasmid
function has made it useful as a plant cloning vector.
The Ti plasmid can be used to shuttle exogenous genes into host plant cells
because the endogenous, tumor‑causing genes of the T-DNA have been inactivated
and foreign genes of interest have been inserted.
CLONING EXPERIMENT
Success of any cloning experiment – depends on being
able to identify the desired gene sequences among the
many different recombinants that may be produced.

DNA fragments + vector

DNA recombinants / clones

Propagation in a host cell

Selection, screening & analysis


the cloned gene
How do we know our
recombinant DNA is
successful?
Selection Transformants : Antibiotic
Resistance
• Transformation produces
bacteria with:
– Religated plasmid
– Religated insert
– Recombinants
• Identify the recombinants
using the antibiotic resistance
– Grow cells with tetracycline so
only cells with plasmid grow,
not foreign DNA only
– Next, grow copies of the
original colonies with ampicillin
which kills cells with plasmid
including foreign DNA
WHAT IS SCREENING?

• The process of identifying one particular


clone containing gene of interest from
among the very large number of others in
the gene library
Gene library – many clones containing gene of
interest

Screening
procedures

To find one particular clone containing gene of


interest
CHARACTERIZATION : METHODS

Restriction mapping – digesting


recombinant DNA with RE
(alone/combination) – construct a
restriction map of the molecule
indicating the cleavage positions &
fragment sizes
Blotting techniques

Methods

Nucleic acid Polymerase Chain


sequencing Reaction (PCR)
RESTRICTION MAPPING
-Restriction
mapping to
confirm that a
plasmid has been
constructed
correctly. -the
orientation of the
clones can be
distinguished by
the restriction
fragment sizes.
SOUTHERN & NORTHERN BLOT
The nucleic acid in lane of of
gel is transferred to a
membrane, bound and the
hybridized with a labeled
nucleic acid probe.

The membrane is the treated


to reveal the bands
produced.

The hybrids, shown as three


bands are visualized by
autoradiography or
chemiluminescence.

Specific RNA species are


detected on Northern blots

The DNA bands on Southern


WESTERN BLOT – USE
ANTIBODY TO DETECT
SPECIFIC PROTEIN
DNA SEQUENCING

Dideoxy DNA sequencing of a


theoretical DNA fragment.
(The “number of
nucleotides” in the gel
analysis refers to nucleotides
added to the primer during
new DNA synthesis.)
POLYMERASE CHAIN REACTION
References

1. Transfection.http://www.biontex.com/en/transfection/
2. https://www.slideshare.net/suvendhudutta/direct-
transformation-method

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