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biotechnology
Cloning
Recap
 What is cloning?
 Generating identical copies of organisms, cells, or replicating nucleic acid
sequences from organisms
 Dolly (the sheep) is a clone, but a natural identical twin is not a clone.
 DNA sequence amplified by growth is a clone,
 How to clone something?
 Application
Cloning Specific Genes
1. Insert the DNA into vector

Gene of interest is inserted into small DNA molecules known as
plasmids, which are self-replicating, extrachromosomal genetic
elements originally isolated from the bacterium, Escherichia coli.

Vectors
Plasmids, or phage
The circular plasmid DNA is opened using the same endonuclease
that was used to cleave the genomic DNA.
Plasmid vector
2. The inserted DNA is joined to the plasmid DNA using another
enzyme, DNA ligase, to give a recombinant DNA molecule.
The new plasmid vector contains the original genetic information
for replication of the plasmid in a host cell plus the inserted DNA
3.The cell
E.coli, yeast
DNA fragment
to be cloned
Vectors
The substance that can serve as carriers to allow
replication of recombinant DNAs.
 Plasmids
 Phage
MCS
Plasmids
Double stranded circles of DNA that can replicate autonomously.
Multiple cloning site. The place where foreign DNA fragments
can be inserted.
Phage λ
 A phage λ virion has a head, which contains
the viral DNA genome, and a tail, which
functions in infecting E.coli host cells.
Viral
Genome
 Advantages over plasmids: They infects
cells much more efficiently than plasmids
transform cells. The yield of clones with
vectors usually higher.
The Cell
E.coli:
 Normal E. coli cells cannot take up plasmid DNA
from the medium. Exposure to high concentration of
certain divalent cations, CaCl2, makes a small fraction
of cells permeable to foreign DNA.
 Each component cell incorporates a single plasmid
DNA molecule.
Inserted Sequence
 Source of Nucleic acid to be cloned:
-DNA directly from organism
-DNA synthesized or amplified in vitro
-Generally a specific sequence.
Two Important Enzymes
 Restriction Enzymes:
cuts the DNA from any organism at specific sequences of a
few nucleotides, generating a reproducible set of fragments.
 DNA Ligases:
insert DNA restriction fragments into replicating DNA
molecules producing recombinant DNA.
first recombinant DNA experiments
 1971 scientists manipulated DNA
and placed them into bacteria
 1972 scientists joined two DNA
molecules from different sources
using the endonuclease EcoRI
(to cut) and DNA ligase (to reseal)
mechanism
How?
Plasmid vector
+
Enzymatically
insert DNA into
plasmid vector
Recombinant
plasmid
Mix E.coli cells with
plasmids
DNA fragment
to be cloned
Bacterial
chromosome
Independent
plasmid
replication
Cell
multiplication
cloning DNA
Introduce the new vector into host
The new vector is inserted back into a host
where many copies of the genetic
sequence are made as the cell grows and
divide with the replicating vector inside.
Isolate the newly-synthesized DNA or the
protein coded for by the inserted gene.
The host may even transcribe and translate
the gene and obligingly produce product
of the inserted gene.
Alternatively, many copies of the DNA gene
itself may be isolated for sequencing the
nucleic acid or for other biochemical
studies.
Library Construction
A library is a collection of different cloned DNAs
from a single source.
 Genomic library – for genome sequencing
 cDNA library – derived from mRNA of a
particular tissue, for isolating specific genes
Revolution in cloning:
(
P
C
R
)
Polymerase Chain Reaction
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What is the Polymerase
Chain Reaction?
 It’s a means of selectively amplifying a
particular segment of DNA.
 The segment may represent a small part of a
large and complex mixture of DNAs:
e.g. a specific exon of a human gene.
 It can be thought of as a molecular
photocopier.
How Powerful is PCR?
 PCR can amplify a usable amount of DNA
(visible by gel electrophoresis) in ~2 hours.
 The template DNA need not be highly
purified — a boiled bacterial colony.
 The PCR product can be digested with
restriction enzymes, sequenced or cloned.
 PCR can amplify a single DNA molecule,
e.g. from a single sperm.
PCR
 Polymerase Chain Reaction (PCR) (PCR cycle)
1. DNA amplified by heating to break hydrogen
bonds, yielding single stranded DNA
2. Short nucleotide sequences act as primers for
DNA replication added
3. Enzyme, DNA polymerase, begins at primers and
synthesizes a DNA strand complementary to the
region between the primers, a process called
"primer extension"
4. Example: 10 cycles = 1,024 copies, 30 cycles =
1,073,741,820 copies
Can I PCR Amplify RNA?
 Not directly — the DNA polymerase
requires a DNA template and will not copy
RNA.
 mRNA can first be copied into cDNA using
reverse transcriptase.
 cDNA is a template for PCR — it need not
be double-stranded.
PCR Animation
Please click here.
Process
Denature
Anneal Primer
Replicate
DNA
1st cycle
2nd cycle
3rd cycle
Applications of PCR
 Mutation testing, e.g. cystic fibrosis.
 Diagnosis or screening of acquired diseases,
e.g. AIDS.
 Genetic profiling in forensic, legal and biodiversity applications.
 Quantitation of mRNA in cells or tissues.
biotechnology
 Biotechnology helps to meet our basic needs.
 Food, clothing, shelter, health and safety
 Improvements by using science
 Science helps in production plants, animals and
other organisms
 Also used in maintaining a good environment that
promotes our well being
A n a l y z i n g
Electrophoresis
c l o n e d
s e q u e n c e s
1. Southern blotting (DNA)
2. Northern blotting (RNA)
3. Western blotting (protein)
First recombinant DNA experiments
 Herbert Boyer discovered a new
technique called gel electrophoresis to
separate DNA/RNA fragments or
proteins
 A current is applied so that the negative
charged DNA/RNA or proteins migrate
towards the positive electrode and is
separated by fragment size
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A Brief Tour of DNA fingerprinting
•Although the structure of DNA is the same
throughout all species of plants, animals and
microorganisms, each individual organism looks
different.
•This is due to the order in which DNA base pairs
are sequenced.
•Not only does this order make you a human
rather than a dog or a daffodil, it also makes each
person unique.
•Sequences of DNA differ from person to person,
but every cell within the same person contains the
same sequence of DNA. So, your hair, blood, skin
and all of the other cells in your body are exactly
the same at the molecular level.
DNA Fingerprint
 This comes in very handy when
police are investigating a crime.
If a person left a strand of hair,
a drop of blood or any other
cells at a crime scene, the police
will know that that person was
there.
But, the human genome
contains about 3 billion base
pairs of DNA. Examining this
large a sequence seems like it
would be tedious, timeconsuming and expensive, so
how is it done?
DNA Fingerprint
 On some human chromosomes, there are sequences of repeated DNA
(9 to 80 base pairs long).
 The number of repeats can vary from about one to thirty and are not
the same from person to person.
 These sequences are called Variable Number of Tandem Repeats
(VNTRs). Within the VNTRs there are sites where an enzyme can cut
the DNA, and the location of these sites also varies from person to
person.
 Cutting with the enzyme will lead to DNA fragments of different
lengths, which are called Restriction Fragment Length Polymorphisms
(RFLPs).
 These DNA fragments can be separated on an agarose gel based on
their size. The RFLPs can be seen by probing using complementary
radioactive DNA, and they are used to compare different samples of
DNA.
DNA Fingerprint
 DNA fingerprinting can be used to identify a
child’s parents. Each child inherits one set of
chromosomes from each parent. This is why
children resemble both of their parents. A child
who has a mom with brown hair and blue eyes and
a dad with blond hair and brown eyes might end
up with brown hair from his mom and brown eyes
from his dad. RFLPs are inherited in the same way,
some from the mother and some from the father.
RFLP
In this example, a family consists
of a mom and dad, two
daughters and two sons. The
parents have one daughter and
one son together, one daughter
is from the mother’s previous
marriage, and one son is
adopted, sharing no genetic
material with either parent.
After amplifying the VNTR
DNA from each member of the
family, it is cut with a
restriction enzyme and run on
an agarose gel.
DNA Fingerprint
•The police use the same analysis to determine the
identity of a person at a crime scene. After collecting
a DNA samples from the scene and any suspects, the
police amplify the VNTRs and digest the DNA with a
restriction enzyme.
•The samples are run on an agarose gel, and the
bands found at the crime scene are aligned with those
of the suspects’.
•DNA fingerprints can do two things, they can either
prove someone’s innocence, or prove their guilt.
The next example shows how DNA fingerprinting can
point to a criminal. DNA samples were taken from a
crime scene, the female victim and two suspects in a
sexual assault case. The victim’s boyfriend was also
tested. The DNA ladders are used to judge the sizes of
the DNA fragments. Control samples are also run, to
ensure that the experiment is done correctly.
p r o d u c t i o n
products of biotech
applications
Agriculture
1. Improved Nutritional Quality
 Milled rice is the staple food for a large fraction of the world's human
population. Milling rice removes the husk and any beta-carotene it
contained. Beta-carotene is a precursor to vitamin A, so it is not surprising
that vitamin A deficiency is widespread, especially in the countries of
Southeast Asia.
 The synthesis of beta-carotene requires a number of enzyme-catalyzed
steps. In January 2000, a group of European researchers reported that they
had succeeded in incorporating three transgenes into rice that enabled the
plants to manufacture beta-carotene in their endosperm.
2. Insect Resistance.
 Bacillus thuringiensis is a bacterium that is pathogenic for a number of
insect pests. Its lethal effect is mediated by a protein toxin it produces.
Through recombinant DNA methods, the toxin gene can be introduced
directly into the genome of the plant where it is expressed and provides
protection against insect pests of the plant.
applications
Agriculture
3. Disease Resistance.

Genes that provide resistance against plant viruses have been
successfully introduced into such crop plants as tobacco, tomatoes,
and potatoes
4. Herbicide Resistance.

Genes for resistance to some of the newer herbicides have been introduced into
some crop plants and enable them to thrive even when exposed to the weed
killer.
applications
Medicine
 Development of novel therapeutic molecules for
medical treatments
 Drug delivery systems
 smart drugs for cancer and autoimmune diseases
gene-based diagnostics and therapies
 pharmaco-genomics and personalised medicine
 health and longevity
public reaction
However, concerns have focused on both
applications and ethical implications:
 Gene therapy experiments have raised the
question of eugenics (artificial human selection)
as well as testing for diseases currently without a
cure
 In agriculture, there is concern about gene
containment and the creation of “super weeds”
(herbicide and/or pesticide resistant weeds)
 Today, fears have focused on genetically
engineered foods in the marketplace and has
resulted in the rapid growth of the organic food
industry