A

Seminar Report
On
Genetic Engineering Techniques
Submitted to the MGSU in partial fulfillment for award of the
degree of
MASTER OF SCIENCE
IN
BIOTECHNOLOGY
SUBMITTED BY:
RASHI GARGI

UNDER SUPERVISION
SHINAM MUKHIJA
SESSION: 2015-2016

MAHARSHI DAYANAND COLLEGE

SRI GANGANAGAR, RAJASTHAN -335001
Phone: 0154-2440756, 2440410 Fax:0154-2444755
www.mdc.edu.in, depingere@mdc.edu.in
Ref. MDC/SGNR/

Date

CERTIFICATE
This is to certify that the Seminar Report entitled Genetic Engineering
Techniques submitted by RashiGargi for the partial fulfillment of the degree of
M.Sc. in the subject of Biotechnology, Maharshi Dayanand College, Sri
Ganganagar (Raj.).

Supervisor

Ms. Shinam Mukhija

Assistant Professor
Department of Biotechnology
Maharshi Dayanand College
Sri Ganganagar, Rajasthan.

INTRODUCTION

Genetic engineering techniques enable modification of the DNA of living

organisms. A variety of editing techniques have been developed since
DNA's structure was first discovered.
FlowchartTARGET
PROCEDURE
HISTORY
LIBRARIES
TECHNIQUES
Targets:

Bacteria are commonly engineered for research purposes.

Typically this is through transformation to add a plasmid
containing a gene of interest, but editing of the chromosome is
also used.

Plants and animals have both been genetically modified for

research, agricultural and medical applications.

genetically modified viruses are also used as viral vectors to

transfer target genes to another organism in gene therapy.

Procedure:
choosing and isolating the gene
Gene combine with promoter/terminator as well as selectable makers
Gene spliced into targets DNA / for animals DNA is inserted in ESCs
Screening

History:

 The bacteria Bacillus thuringiensis was first discovered in 1901 as

the causative agent in the death of silkworms.
Due to these insecticidal properties the bacteria was used as an
biological insecticide, commercially developed in 1938.
The cry proteins were discovered to provide the insecticidal activity
in 1956 and by the 1980s scientists had successfully cloned the
gene coding for this protein and expressed it in plants.
Libraries:
Target genes can be cloned from a DNA segment after the creation
of a DNA library.
The libraries generally cover the organism's genome multiple times
and its size will depend on how large the genome is.
Techniques:
Blotting Techniques
Gel Retardation
DNA Fingerprinting & DNA Footprinting
Chromosome Walking
Blotting Techniques: Visualization of specific DNA , RNA & protein among
many thousands of contaminating molecules requires the convergence of
number of techniques which are collectively termed BLOT transfer .
Types of blotting techniques:
1) Southern blotting (to detect DNA)
2) Northern blotting (to detect RNA)
3) Western blotting (to detect protein)

Southern Blotting:
In 1975 Edward Southern developed this technique that is widely
used to detect fragments of DNA .
This requires
1) Separation of DNA or DNA fragments by

agarose gel

electrophoresis .
2) DNA fragments are blotted onto a strip of nitrocellulose or a nylon
membrane.
3) Identification by hybridization with a labeled ,complementary
nucleic acid probe.
Southern blot a method for transferring DNA from an agarose gel to
nitrocellulose filter , on which the DNA can be detected by suitable
probe ( eg : complementary DNA or RNA ) .

Procedure:
The DNA sample is digested by restriction endonucleases, producing
small fragments & that are amenable for analysis.
Fragments are seperated by agarose gel electrophoresis or PAGE.
The mobility of nucleic acids in agarose gels is influenced by
agaroseconcentration, molecular size & molecular conformation of
the nucleic acid.
Agarose concentration of 0.3 2 %are most effective for nucleic acid
separation.
Like proteins nucleic acids migrate at rate that is inversely
proportional to the logarithm of their molecular weight.
Separated

nucleic

acids

are

visualized

by

fluorescent

dye

ethidiumbromide.
The agarose gel is soaked in a solution of dye & washed for remain
excess dye.
illumination of the rinsed slab with UV light reveals red orange
stains where nucleic acids are located .
Ethidium bromide stains both single & double stranded nucleic
acids, the fluorescence is much greater with double stranded
molecules.
The electrophoresis can be performed with dye incorporated in the
gel &buffer.
This has the advantage that the gel can be illuminated with UV light
during electrophoresis to view the extent of separation.

 The mobility of DNA may be reduced by 10 -15 % in the presence of

ethidium bromide.
Ethidium bromide must be used with great care as it is a potent
mutagen.
Gloves should be worn at all times while using the dye solutions or
handling gels.
Blotting:
Transfer of DNA from gel to nitrocellulose membrane done by
1) Weak acid treatment to depurinate & fragment the DNA, thus
make it smaller & easier to elute from the gel followed by
2)

Denaturate

with

strong

base&neutralisation(hydrolyzesphosphodiester back bone at depurinated

sites )
Single strands bind to membranes more efficiently
A buffer is used to facilitate the transfer.
Original methods of transfer relied on capillary action.
Vaccum or preassure systems can be used to speed the transfer.
Faster & more efficient transfer is afforded by the use of an
electroblotter.
Electroblotting process is usually completes in 1-4 hours.
Applications:
Southern blots are used in gene discovery, mapping, evolution &
development studies, diagnostics & forensics.
Deletions / insertions.
pointmutations / polymorphisms.

 Structural rearrangements.
Allow

for

determination

of

molecular

weights

of

restriction

fragments.
Presence of particular bit of DNA in the sample.
Northern Blotting- Applications
A standard for direct study of the gene expression at the level of
mRNA.
Detection of mRNA transcript size.
Study of RNA splicing can detect alternatively spliced transcripts.
Study RNA half life
Disadvantages:
Time consuming procedure.
RNA samples can be degraded by RNases.
Use of radioactive probes.
Detection with multiple probes is a problem.
Western Blotting- Applications

The confirmatory HIV test employs a western blot to detect anti

HIV antibody in a human sample.

Proteins from known HIV infected cells are separated & blotted on
a membrane then the serum to be tested is applied in the
primary antibody incubation step.

Free antibody is washed away & a second anti human antibody

linked to an enzyme signal can be added.

The stained bands then indicate the proteins to which the patient
serum contains antibody.

Western blot is also used as definitive test for bovine spongiform

encephalopathy.

(mad cow disease )

Some forms of Lyme disease testing employs western blotting.

Gel Retardation:
Gel retardation (band shift) technique offered the combination of
high analytical power and technical Simplicity.
This procedure is based on the observation that the formation of
protein-nucleic complexes generally reduces the electrophoretic
mobility of the nucleic acid component in the gel matrix.
The factors which contribute most to the resolution of the complex
from the naked nucleic acid are the gel pore size, the relative mass
of protein compared with nucleic acid, and changes in nucleic acid
conformation (bending) induced by binding.
The consequences of induced bending on the mobility of doublestrand DNA fragments are similar to those arising from sequencedirected bends, and the latter can be used to help characterize the
angle and direction of protein-induced bends.
Whether a complex formed in solution is actually detected as a
retarded band on a gel depends not only on resolution but also on
complex stability within the gel.
This is strongly influenced by the composition and, particularly, the
ionic strength of the gel buffer.
Chromosome Walking:

Chromosome walking is a method of positional cloning used to

find, isolate, and clone a particular allele in a gene library.

Chromosome Walking was developed by Welcome Bender, Pierre

Spierer, and David S. Hogness in the Early 1980's.

There are nearly half a dozen positional cloning tests that are
done prior to a chromosome walk.

Each clone in the cosmic library has a DNA insert of 50 KB.

The walking starts at the closest gene that has already been
identified, known as a marker gene.

A
pplication:
This technique can be used for the analysis of genetically
transmitted diseases, to look for mutations.
Chromosome Walking is used in the discovery of single-nucleotide
polymorphism of different organisms.
Disadvantages:

There

is

a limitation to the

speed

of

chromosome

walking because of the small size of the fragments that are to be

cloned.

Another

limitation

is

the

difficulty

of

walking

through

the repeated sequence that are scattered through the gene.

If the markers were too far away, it simply was not a viable
option.

Additionally, chromosome walking could easily be stopped by

unclonable sections of DNA.

A solution to this problem was achieved with the advent of

chromosome jumping (Marx, 1989), which allows the skipping of
unclonable sections of DNA.

DNA footprinting: Aim- to assess whether a given protein binds to a

region of interest within a DNA molecule.
Take the genomic DNA
Polymerase chain reaction (PCR) amplify and label region of interest
that contains a potential protein binding site

Take a sample as
control
Addprotein of interest to a
portion of the labeled
template DNA

Add a cleavage agent to both portions of DNA template

Run

both

samples

electrophoresis.

side

by

side

on

polyacrylamide

gel

 The portion of DNA template without protein will be cut at random

locations, and thus when it is run on a gel, will produce a ladder like
distribution.
The DNA template with the protein will result in ladder distribution
with a break in it, the "footprint", where the DNA has been protected
from the cleavage agent.

DNA Fingerprint:
DNA (deoxyribonucleic acid) represents the blueprint of the human
genetic makeup.
It exists in virtually every cell of the human body and differs in its
sequence of nucleotides (molecules that make up DNA, also
abbreviated by letters, A, T, G,C; or, adenine, thymine, guanine, and
cytosine, respectively).
The human genome is made up of 3 billion nucleotides, which are
99.9% identical from one person to the next.
The 0.1% variation, therefore, can be used to distinguish one
individual from another.
It is this difference that can be used by forensic scientists to match
specimens of blood , tissue, or hair follicles to an individual with a
high level of certainty.
The complete DNA of each individual is unique, with the exception
of identical twins.
A DNA fingerprint, therefore, is a DNA pattern that has a unique
sequence such that it can be distinguished from the DNA patterns of
other individuals.
DNA fingerprinting is also called DNA typing.
DNA fingerprinting is based on DNA analyzed from regions in the
genome that separate genes called introns.
They are spliced out during processing of the messenger RNA, which
is an intermediate molecule that allows DNA to encode protein.

 This is in contrast to DNA analysis looking for disease causing

mutations, where the majority of mutations involve regions in the
genes that code for protein called exons.
DNA fingerprinting usually involves introns because exons are much
more conserved and therefore, have less variability in their
sequence.