Genetic engineering and the Human Genome Project

roachavocadoBiotechnology

Dec 14, 2012 (4 years and 9 months ago)

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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