Genetic engineering and the Human Genome Project

Biotechnology

& Recombinant DNA

What is
biotechnology?


Using living
microorganisms or
cell components to
make products


Often via genetic
engineering and
using recombinant
DNA

Recombinant DNA technology:


Insertion or
modification of genes
to produce desired
proteins.

What is
recombinant DNA?


Inserting foreign DNA
into a bacterial cell


Use
restriction
enzymes
, plasmids,
ligase and bacterial
host


Restriction enzymes


Defense against viruses


Clone gene or make
gene product

How is recombinant DNA made?


Recall: happens
naturally


Transposons


Researchers can also
produce it


Use bacterial plasmids or
viruses (bacteriophages)


Uses


Bacteria make insulin


yeast help make
components of Hepatitis
B vaccine

What are restriction enzymes?


Naturally occur
in bacteria


Bacteria use
these to
combat viral
infection


Bacteria DNA
uneffected
because some
is methylated


Sequence
-
specific


Restriction
enzymes


Cloning
animation


This can be used as a vector…

What is a vector?


Different from a disease
vector!


plasmid animation


Plasmid or virus


Used to insert DNA into host
cell


Must be able to self
-
replicate!


Must be small so not fragile



Both types of vectors can
allow researchers to clone
DNA


But there’s another approach
to DNA amplification…

PCR: making copies of DNA

What is PCR?

Figure 9.4 (1 of 2)

Stands for _____________


Need primers of about 20 bp
to start

DNA polymerase doesn’t
start reaction, only
lengthens

Primers recognize
regions which flank
target gene


Ingredients

primers

DNA polymerase from
TAC (
Thermus aquaticus
)

Individual nucleotides


Can you go over that one
more time? …
PCR animation

What is PCR?

Figure 9.4 (2 of 2)

Stands for _____________


Need primers of about 20 bp
to start

DNA polymerase doesn’t
start reaction, only
lengthens

Primers recognize
regions which flank
target gene


Ingredients

primers

DNA polymerase from
TAC (
Thermus aquaticus
)

Individual nucleotides


Can you go over that one
more time? …
PCR animation

Why would you use PCR?


Detect small amounts
of DNA


Can you think of
examples?


Get in groups and
discuss!


Forensics


Infectious agents


Gene mapping


Human Genome
Project


Taxonomy and
systematics studies


Cancer and study of
other human diseases


Sequencing of rRNA
and mRNA via cDNA


DNA fingerprinting

Let’s take a brief look at some of
these…

What is gel electrophoresis?


Sorting DNA segments by size


DNA fingerprinting


Restriction enzymes create restriction fragment length
polymorphisms (RFLPs)

Gel
electrophoresis
animation

What is Southern
blotting?


After gel
electrophoresis


Filter paper blots DNA
off


Radioactive probes
added


Autoradiography
pinpoints sequence


Southern Blot
animation


The scientific applications


Understanding of
DNA


Sequencing
organisms' genomes


DNA fingerprinting
for identification



Figure 9.17

Why
E. Coli
is used!


Used because it is easily grown and its
genomics are known


Need to eliminate endotoxin from products


Cells must be lysed to get product

Inserting Foreign DNA into Cells


DNA can be inserted into a cell by


Transformation


Electroporation


Protoplast fusion


Microinjection


Gene gun

What is RNA Interference (RNAi)?

Figure 9.14

RNAi animation


Cloning

How do researchers get the DNA they
want to clone?


Gene libraries are made
of pieces of an entire
genome stored in
plasmids or phages.


cDNA is made from
mRNA by reverse
transcriptase.


Synthetic DNA is made
by a DNA synthesis
machine.

So what’s the problem?


Fine for prokaryotic DNA


Problem with eukaryotic
DNA…


Eukaryotic DNA has
introns…


Must make
complementary DNA
(cDNA)


Use reverse trasncriptase


cDNA animation


Now DNA can be inserted…

What do they do with the cloned DNA?


Lots of different things!

Is this the only way to “look” at DNA?


No
—
we can sequence it, too!


Random shotgun sequencing


Start with a whole genome or a large piece of the DNA (a BAC).


BAC
-
bacterial artificial
choromosme


Shear the DNA into many different, random segments.


Sequence each of the random segments Put the pieces back together
in original order

What are bioreactors?


Using bacteria to
produce gene products


Insulin: diabetes


Human growth
hormone


Cellulase (break down
cell wall for animal
feed)


Factor VIII: hemophilia


What are plant GMOs?


Genetically modified organisms


Transgenic plant or animal


Bioreactors filled with these


Plants


Cotton, corn, potato to make pest resistant


Soybeans resistant to common herbicide


Some corn, cotton are herbicide and pest resistant


Could produce human hormones, clotting factors, antibodies on seeds
in future


What are animal GMOs?


Foreign genes into
embryos


produce animals that
manufacture human
hormones, etc. = gene
pharming


Blood clotting factor
goats


Sheep milk with
human alpha
-
1
-
antitrypsin (used to
treat heritable
emphysema)


1997, Dolly


Since then, cloned
sheep, cows, goats, mice


Humans: moratorium

What is gene
therapy?


Insertion of genetic
material into human
cells to treat a disorder


Use retrovirus to insert
normal gene into cell


Healthy genes to make
up for faulty genes


Severe combined
immunodeficiency syndrome


1990, girl received normal
gene in white blood cells


Using genes to treat
other illnesses