Gene Cloning (Genetic technology,Biotechnology).pdf

Gene Cloning

1. 6.1 Gene Cloning – DNA manipulation
6.2 Biotechnology – Applications: medicine, agriculture & industry
 Unit 6
Genetic Technology

2. 13-2 Is selective breeding the same as genetic engineering?

3. 13-3
Dolly

5. Genetic Technology
- Concepts
Recombinant DNA technology, genetic engineering, and
biotechnology have revolutionized medicine and agriculture.
Genetic modifications of plants have resulted in herbicide-
and pest-resistant crops, and crops with improved nutritional
value; similarly, transgenic animals are being created to
produce therapeutic proteins and to protect animals from
disease.
Applications of recombinant DNA technology and genomics
have become essential for diagnosing genetic disorders,
determining genotypes, and scanning the human genome to
detect diseases.
Gene therapy by transfer of cloned copies of functional alleles
into target tissues is used to treat genetic disorders.

6. 11-6
Genetic Modification of
Organisms
It is now possible to clone genes and move them from one
organism to another.
called gene cloning
DNA sequences can be altered (mutated) to generate a
desired change.
The new DNA is called recombinant DNA (rDNA).
Once the DNA is transferred, the new host cell begins to
make the new DNA and produce the new proteins.
Organisms that contain recombinant DNA are called
“genetically modified organisms” (GMO’s).
Usually involves bacteria or viruses that will make large
amounts of the protein of interest

7. DNA Technology
Our understanding of genes and the base sequences of
DNA has facilitated the development of genetic
engineering.
Genetic engineering can be used to clone (copy) genes
from an organism and use that gene to alter the genome
of another organism.
Genetic engineering forms recombinant DNA technology
Use of laboratory techniques to isolate and manipulate
fragments of DNA
 Recombinant DNA (rDNA) contains DNA from 2 or
more different sources.
Once inside a host cell, recombinant molecules are replicated to
produce identical copies or clones

8. Recombinant DNA Technology
Gene cloning (= recombinant DNA technology):
A vector, such as a bacterial plasmid, serves as a
carrier for the foreign gene.
Restriction enzymes are used to cut the plasmid and
splice in the foreign gene.
DNA ligase seals the gene into the plasmid.
Scientific Revolution

9. A gene transfer experiment occurs in four stages

1.Cleaving DNA
cutting the source and vector DNA

2.Producing recombinant DNA
placing the DNA fragments into vectors and then transferring the
DNA into the target cells

3.Cloning
introducing DNA-bearing vectors into target cells and then
allowing the target cells to reproduce

4.Screening
selecting the particular infected cells that have received the gene
of interest
…. a Scientific Revolution
Scientific Revolution

10. A Scientific Revolution
Genetic engineering is moving genes from one
organism to another
the first stage in a genetic engineering experiment is
to chop up the source DNA and obtain a copy of the
gene you want to transfer
restriction enzymes bind to specific short sequences
on the DNA and make a specific cut
the sequence is symmetrical
the cut generates DNA fragments that are “sticky” because
the incision made by the restriction enzyme is made to the
side
restriction enzymes are the basic tools of genetic engineering


Scientific Revolution

11. 11-11
How to Clone a Gene
Cut the gene of interest out of the
chromosome using restriction enzymes.

12. 11-12
How to Clone a Gene
Splice the fragment
containing the gene into a
carrier molecule, usually a
bacterial plasmid.

13. 11-13
How to Clone a Gene
Insert the plasmid with the fragment into the bacterial
cells.

14. 11-14
How to Clone a Gene
Each time the bacterial cells divide, many copies of
the gene will be made.

15. 15
Gene cloning - used to produce
large amounts of a gene or its
protein product

16. 11-16
Genetically Modified Organisms
Genetically modified organisms have been used
to:
Make human insulin
Generate “Golden rice”
Make human growth hormone
For bioremediation (the use of living organisms to remove
toxins from the environment)
Generate crops that supply developing nations with
nutrients not normally found in their native plants
Generate crops that can manufacture medicines to treat
disease
Generate crops that are resistant to herbicides or that
make their own insecticides

17. 11-17
Gene Therapy

Recombinant DNA technology can be used
to administer gene therapy.
Gene therapy involves manipulating genes in
order to cure or treat a genetic disease.
Gene therapies must be specifically designed for
each situation.
If the mutant gene is not functional, then a
functional gene must be inserted.
If the mutant gene is overactive, then it must be
deleted or altered.
Usually involves mutating the part of the gene that
controls its activation

18. Electrophoresis
Technique that is used to
separate macromolecules,
such as DNA and proteins,
on a gel
Can be used to separate
molecules based on their
charge, size/length, and
mass

18
1
2
3
Higher-mass molecules
Lower-mass molecules
Samples
Gel
–
+
–
+
Load samples
of DNA fragments
into wells at the
top of the gel.
Apply an
electric field.
Wait additional
time.
Each band is a group of
DNA fragments with the
same mass.
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19. Polymerase Chain Reaction
• Polymerase chain reaction (PCR) is a technique
that amplifies (quickly makes multiple copies of)
a DNA sequence.

PCR requires primers that start the process of
DNA replication on the DNA strand.

PCR also requires DNA polymerase to
synthesize the new DNA strand.
To be continued …..Lecture #2

20. Reading list:
1. Brooker et al.2008, Ch 20
2. Hyde 2009, Ch 12
13-20