Lecture 24: Biotechnology

Dr. Kim Green

kngreen@uci.edu

Wednesday 25th November 2015

Chapter 20: Pages 408- 433

Biotechnology is the
manipulation of organisms
or their components to
make useful products
The applications of DNA
technology affect
everything from agriculture,
to criminal law,
to medical research

The DNA Toolbox

Sequencing of the human genome was completed
by 2007
DNA sequencing has depended on advances in
technology, starting with making recombinant DNA
In recombinant DNA, nucleotide sequences from
two different sources, often two species, are
combined in vitro into the same DNA molecule

How is the word clone defined?

1) A lineage of genetically identical individuals or cells
2) A single individual organism that is genetically identical to another individual
3) To make one or more genetic replicas of an individual or cell

What does it mean to clone a gene?

The production of multiple copies of a gene

How do you
clone a gene?

The production of multiple

copies of a gene
(aka: gene cloning)

Why would you want

to clone a gene?

Using Restriction Enzymes to Make Recombinant

DNA
Bacterial restriction enzymes cut DNA molecules at specific
DNA sequences called restriction sites
A restriction enzyme usually makes many cuts, yielding
restriction fragments
The most useful restriction enzymes cut DNA in a staggered
way, producing fragments with sticky ends that bond with
complementary sticky ends of other fragments

DNA ligase is an
enzyme that seals the
bonds between
restriction fragments

Cloning a Eukaryotic Gene in a Bacterial Plasmid

In gene cloning, the original plasmid is called a
cloning vector
A cloning vector is a DNA molecule that can
carry foreign DNA into a host cell and replicate
there

Amplifying DNA in Vitro: The Polymerase Chain

Reaction (PCR)
The polymerase chain reaction, PCR, can
produce many copies of a specific target
segment of DNA
A three-step cycleheating, cooling, and
replicationbrings about a chain reaction that
produces an exponentially growing population
of identical DNA molecules

 Allows us to study a specific gene/DNA sequence,

even though the actual amount of DNA in a cell is
very very small
Can also be used to amplify a gene which is then
inserted into an expression vector (i.e. Bacterial
plasmid).
Needs template DNA, dNTPs, water and DNA
polymerase + primers.

PCR is widely used in all sorts of labs

around the world.
Fragments of ancient DNA from a 40,000 year old frozen wooly
mammoth

DNA from fingerprints or from tiny amounts of blood, tissue, or

semen found at crime scenes

DNA of viral genes from cells infected with viruses that are
difficult to detect, such as HIV

Gel Electrophoresis
Gel acts as a molecular sieve to
separate DNA molecules on the
basis of size, electrical charge, and
other physical properties

Is DNA negatively or positively

charged?

So far we have been talking about gene

cloning and the manipulation of individual
pieces of DNA, etc.
Now, onto the reproductive cloning of
mammals..

In 1997, a lamb was

cloned from an adult
sheep by nuclear
transplantation
Dolly was euthanized in 2003
due to early development of an
arthritic condition as well as a
lung disease usually observed
only in much older sheep.
Suggested reprogramming of
original transplanted nucleus
was incomplete
Also demonstrates that cloned
animals do not always end up
identical to donor

What about mitochondrial DNA?

Problems Associated with Animal Cloning

In most nuclear transplantation studies, only a
small percentage of cloned embryos have
developed normally to birth
Many epigenetic changes, such as acetylation of
histones or methylation of DNA, must be reversed
in the nucleus from a donor animal in order for
genes to be expressed or repressed appropriately
for early stages of development

Stem Cells of Animals

A stem cell is a relatively

unspecialized cell that can
reproduce itself indefinitely and
differentiate into specialized cells
of one or more types

Stem cells isolated from early

embryos at the blastocyst stage
are called embryonic stem cells;
these are able to differentiate into
all cell types

The adult body also has stem

cells, which replace
nonreproducing specialized cells

Stem cells:
Embryonic stem cells
Vs.
Adult stem cells
One major goal of stem cell
research is to provide functional
cells for treating human diseases.
Alzheimers
Spinal cord injury
Insulin-producing pancreatic cells
Immuno system
This all falls under therapeutic
cloning.

Induced Pluripotent Stem (iPS) Cells

Researchers can treat differentiated

cells, and reprogram them to act
like ES cells

Researchers used retroviruses to

induce extra copies of four stem cell
master regulatory genes to produce
induced pluripotent stem (iPS) cells

iPS cells can perform most of the

functions of ES cells

iPS cells can be used as models for

study of certain diseases and
potentially as replacement cells for
patients

The practical applications:

1) Diagnosis of diseases
2) Human gene therapy
3) Pharmaceutical products
4) Protein production
5) Forensic evidence and genetic profiles
6) Environmental clean up
7) Agriculture applications
8) list is almost endless.