BIOTECHNOLOGY

Sahib dino 28575(bs physics 5th semester )

GENE CLONING

1. A fragment of DNA, containing the gene to be cloned,

is inserted into a circular DNA molecule called a
vector, to produce a recombinant DNA molecule.
2. The vector transports the gene into a host cell, which
Chapter 1
is usually a bacterium, although other types of living
cell can be used.
3. Within the host cell the vector multiplies, producing
numerous identical copies, not only of itself but also of
the gene that it carries.
4. When the host cell divides, copies of the recombinant
DNA molecule are passed to the progeny and further
vector replication takes place.Analysis
place. are Important
5. After a large number of cell divisions, a colony, or
clone, of identical host cells is produced. Each cell in
the clone contains one or more copies of the
recombinant DNA molecule; the gene carried by the
recombinant molecule is now said to be cloned.ne
cloned.
Cloning and DNA
Gene Cloning: The basic steps in gene cloning.
Fig 1.1
The advent of Gene Cloning and the Polymerase
chain reaction (PCR)

1971-1973 new methodologies were developed,

referred to as Recombinant DNA technology or
Genetic Engineering and Gene Cloning.
These modern techniques spawned the
biotechnology.
In 1985 Kary Mullis invented the PCR , by using
the thermo cycler.
PCR (Polymerase Chain Reaction)

 PCR- very different from gene cloning (series of

manipulations involving living cells).
 Carried out in a thermo cycler
 Basic steps in a PCR experiment are:
1. Denaturation of template strand
2. Annealing of oligonucleotides primers
3. Synthesis of new chain by Taq polymerase
At the end of 1st cycle, two dsDNA molecule (8 strands)
are formed.
Now, begins a second cycle of denaturation-annealing-
synthesis.
By the end of 30 cycles, we get over 130 million new
double-stranded molecules.
Fig 1.2: The basic steps in the polymerase chain reaction
Limitations of PCR:

1. Sequences of the either sides of the gene of

interest (annealing sites) must be known
So, PCR cannot be used to isolate genes that have
not been studied before that has to be done by
gene cloning.
2. PCR can amplify only a limited length of
sequence, i.e. five Kilo bases (kb)
So, Cloning must be used to get intact version of
long genes.
Important applications of PCR:

 For an unknown gene sequence, primers can be

determined from equivalent gene.
 It is very easy to isolate or detect genes whose
sequences are already known, by using PCR.
 PCR also involves the use of primers, specific for
the DNA of a disease-causing virus
 PCR is tremendously sensitive, yields detectable
amounts of DNA .
Dolly the sheep
 The first cloned was Dolly, a sheep who was born in
1996. 
 To create Dolly, the nucleus was removed from a
donor egg cell.
 they were shocked to fuse. They were shocked again
to start division. 
 The cells were allowed to divide for several days until
an early embryonic stage was reached, before being
implanted in a surrogate mother.
Gene Therapy
 Gene therapy is the introduction of genes into
existing cells to prevent or cure a wide range of
diseases.
 This can be accomplished by:
 Replacing a mutated gene that causes disease with a
healthy copy of the gene.
 Inactivating, or “knocking out,” a mutated gene that is
functioning improperly.
 Introducing a new gene into the body to help fight a
disease.
Advantages
 It is a possible cure for heart disease, AIDS and
cancer.

 It gives someone born with a genetic disease a

chance to life.

 It can be used to eradicate diseases from the future

generations.
Disadvantages
 Long lasting therapy is not achieved by gene therapy;
Due to rapid dividing of cells benefits of gene therapy is
short lived.
 Immune response to the transferred gene stimulates a
potential risk to gene therapy.
 Viruses used as vectors for gene transfer may cause
toxicity, immune responses, and inflammatory reactions
in the host.
 Disorders caused by defects in multiple genes cannot be
treated effectively using gene therapy.
Plant tissue Culture
 Plant tissue culture is a technique of growing
plant cells, tissues, organs, seeds or other
plant parts in a sterile environment on a
nutrient medium.
Plant Tissue Culture
Applications
 The commercial production of plants used as potting,
landscape, and florist subjects

 To conserve rare or endangered plant species.

 To screen cells rather than plants for advantageous

characters, e.g. herbicide resistance/tolerance.

 Large-scale growth of plant cells in liquid culture in

bioreactors for production of valuable compounds, like
plant-derived secondary metabolites and recombinant
proteins used as biopharmaceuticals.
 To cross distantly related species by protoplast fusion
and regeneration of the novel hybrid.

 To produce clean plant material from stock infected by

viruses or other pathogens.

 Production of identical sterile hybrid species can be

obtained.
Biotechnology Products
 Products Which are Manufactured By Recombinant
DNATechnology(Produced By Biotechnology).
 Examples:
 Antibiotics
 Transgenic plants
 Vaccines
 Genetically modified organisms