Transcript Genetic Engineering_AP Bio

Ch 20: DNA Technology
Introduction
• The mapping and sequencing of the human genome has
been made possible by advances in DNA technology.
• Human genome project
• We are now developing techniques for making
recombinant DNA
• Genes from two different sources - often different
species - are combined into the same molecule.
• DNA technology has launched a revolution in
biotechnology.
• The manipulation of organisms or their components to
make useful products.
• DNA technology is now applied in areas ranging from
agriculture to criminal law, but its most important
achievements are in basic research.
Recombinant DNA
• Recombinant DNA
– Taking DNA from two sources and
combining then into one molecule.
– Occurs naturally in viral transduction,
bacterial transformation, and conjugation
• Biotechnology (genetic engineering)
– Engineering genes in the Lab
Many tools and techniques have been
developed to manipulate and engineer genes.
1. Restriction Enzymes
2. Gel Electrophoresis
-Restriction Fragment Polymorphisms
3. DNA Probe
4. Polymerase Chain Reaction
5. Complementary DNA
Restriction Enzymes
• Discovered in the 1960s
• Extracted from bacteria
– Used to fend off bacteriophages
– Appear to serve a host-defense role
– Protect own DNA by methaylation of adenines &
cytosines
• REs cut DNA at specific sites called recognition
sequences or sites.
– Leaving fragments of DNA
• Scientists have isolated 100s of REs
– Named for the bacteria in which they were found.
• EcoRI
• BamHI
• HindIII
Naming
Restriction enzymes are named based on the bacteria in which
they are isolated in the following manner:
E
Escherichia
(genus)
co
R
I
coli
(species)
RY13
(strain)
First identified Order ID'd in bacterium
Enzyme
Ava I
Organism from which derived
Target sequence
(cut at *)
5' -->3'
Anabaena variabilis
C* C/T C G A/G G
Bacillus amyloliquefaciens
G* G A T C C
Bgl II
Bacillus globigii
A* G A T C T
Eco RI
Escherichia coli RY 13
G* A A T T C
Eco RII
Escherichia coli R245
* C C A/T G G
Hae III
Haemophilus aegyptius
G G * C C
Hha I
Haemophilus haemolyticus
G C G * C
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Hpa I
Haemophilus parainflenzae
G T T * A A C
Kpn I
Klebsiella pneumoniae
G G T A C * C
Mbo I
Moraxella bovis
*G A T C
Mbo I
Moraxella bovis
*G A T C
Pst I
Providencia stuartii
C T G C A * G
Sma I
Serratia marcescens
C C C * G G G
Streptomyces stanford
G A G C T * C
Bam HI
SstI
• The restriction sites are often a symmetrical
series of four to eight bases on both strands
running in opposite directions.
• If the restriction site on one strand is 3’CTTAAG-5’, the complementary strand is 5’GAATTC-3’.
• Because the target sequence usually occurs (by
chance) many times on a long DNA molecule, an
enzyme will make many cuts.
• Copies of a DNA molecule will always yield the same
set of restriction fragments when exposed to a
specific enzyme.
CLIP
• Restriction enzymes cut
covalent phosphodiester
bonds of both strands
• often in a staggered way
creating single-stranded
ends, sticky ends.
• These extensions will
form hydrogen-bonded
base pairs with
complementary singlestranded stretches on
other DNA molecules cut
with the same restriction
enzyme.
•These DNA fusions can be made permanent by DNA
ligase which seals the strand by catalyzing the formation
of phosphodiester bonds.
Must use same
restriction
enzyme on both.
Setting up a simple restriction digestion:
1. DNA: Reliable cleavage by restriction enzymes requires DNA that is free
from contaminants such as phenol or ethanol. Excessive salt will also
interfere with digestion by many enzymes, although some are more tolerant
of that problem.
2. An appropriate buffer: Different enzymes cut optimally in different buffer
systems, due to differing preferences for ionic strength and major cation.
When you purchase an enzyme, the company almost invariably sends along
the matching buffer as a 10X concentrate.
3. The restriction enzyme!
Restriction
Enzyme
animation
Animation #2
Cloning a Gene
Bacteria DNA that has DNA
from another organism
spliced in to it.
Gel Electrophoresis
• Method of rapidly analyzing and comparing
genomes.
Gel Electrophoresis
Restriction Fragment Length Polymorphisms
(RFLPs)
• The restriction pattern is different for every
organism.
• This is why you get different banding patterns.
Agar is an unbranched polysaccharide obtained from the cell walls of some species of red algae or
seaweed
DNA fragments are
visualized by staining
with ethidium bromide.
This fluorescent dye
intercalates between
bases of DNA and RNA
Rate of movement
depends on size,
electrical charge, and
other physical properties
of the macromolecules.
• Separation depends mainly on size
(length of fragment) with longer
fragments migrating less along the
gel.
• Because DNA has
a negative charge,
it moves toward
the opposite side.
• Smaller
fragments move
greater distances
Simulation
We can tie together several molecular techniques to
compare DNA samples from three individuals
We start by adding the restriction enzyme to each of the three
samples to produce restriction fragments.
• We then separate the fragments by gel electrophoresis.
• Southern blotting (Southern hybridization) allows us to transfer the
DNA fragments from the gel to a sheet of nitrocellulose paper, still
separated by size.
• Southern blotting -method in molecular biology of enhancing the
result of an agarose gel electrophoresis by marking specific DNA
sequences
• This also denatures the DNA fragments.
Animation
• Bathing this sheet in a solution containing our probe allows the probe to
attach by base-pairing (hybridize) to the DNA sequence of interest and
we can visualize bands containing the label with autoradiography.
Polymerase Chain
Reaction (PCR); Making copies
• Common method of
creating copies of specific
fragments of DNA
• PCR rapidly amplifies a
single DNA molecule into
many billions of
molecules
Animation
Complementary DNA (cDNA)
• When scientists
clone a human
gene in a
bacterium, the
introns present a
problem.
• Bacteria lack
introns and have
no way to cut
them out.
Animation
Complementary DNA (cDNA)
• In order to clone a
human gene in a
bacterium, the introns
need to be removed.
• The genes is allowed to
be transcribed and
fully processed into
mRNA.
• Reverse transcriptase
is added to the mRNA
and DNA copies are
made.
• The DNA made by this
process is called cDNA.
DNA probe; DNA Tagging
• A DNA probe is a radioactively labeled
single strand of nucleic acid molecule
used to tag a specific sequence in a DNA
sample.
• The probe bonds to a complementary
sequence wherever it occurs.
• Can identify genetic defects
Animation
Gene Therapy
Cloning
Genetically Modified Organisms
Clip
Transgenic Organisms
Transgenic Tobacco, from
1986. This is an ordinary
photographic image of a tobacco
plant engineered to express a
firefly gene which produces
luciferase.
CLIP
72
Plasmids as vectors
• Lab on Fri
• Animation
71
Golden Rice
23 times more Vitamin A
Called a Transgenic Organism
•Researchers use recombinant
DNA technology to analyze
genetic changes.
•They cut, splice together, and
insert the modified DNA
molecules from different
species into bacteria or another
type of cell that rapidly
replicates and divides.
•The cells copy the foreign DNA
right along with their own DNA.
•An example of this is the gene
for human insulin inserted into a
bacterium. This is how human
insulin is mass produced.
•Not only does genetic engineering have
applications in medicine and the
environment, it also has uses in industry
and agriculture.
•Sheep are used in the production of alpha1 antitrypsin, which is used in the
treatment of emphysema.
•Goats are also producing the CFTR protein
used in the treatment of cystic fibrosis.
In the plant world, the buds of cotton
plants are vulnerable to worm
attacks. The buds of a modified
cotton plant resist these worms,
resulting in increased cotton
production.
These gene insertions are
ecologically safer than pesticides.
They affect only the targeted pest.
Plant biologists have used DNA
technology to produce plants
with many desirable traits.
These include increased disease
resistance, herbicide resistance,
and increased nutritional
content.
Scientists today have developed genetically
altered bacteria.
Among them are strains of bacteria that
eat up oil spills
manufacture alcohol and other
chemicals
process minerals.
There is concern about possible risks to the
environment and the general population as
genetically engineered bacteria are
introduced.