Download BIOTECHNOLOGY

GENETIC MODIFICATIONS
 Genetic engineering: altering the
sequence of DNA
 Ideas established in early 70's by
2 American researchers, Stanley
Cohen (worked with plasmids) and
Herbert Boyer (restriction
endonucleases)
 Initially had no commercial
applications for their experiments,
but things changed quickly.
 In 1976 Boyer cofounded
Genetech, first biotech company
to go public on the stock market.
 1978: somatostatin became the first human
hormone produced by this technology
 Other examples:
 Insulin: over 90% diabetics are reliant on human
insulin supplied by bacteria.
 Somatropin: used to treat human growth
deficiency, from dwarfism, Turner's syndrome, also
used for AIDS-associated wasting syndrome now
BIOTECHNOLOGY
 Biotechnology involves the manipulation of DNA and
protein synthesis.
 Molecular biologists analyze and alter genes and
their respective proteins
Examples
 Genetic screening: scanning for genetic mutations
 Gene therapy: the alteration of a genetic sequence
in an organism to prevent or treat a genetic disorder
by creating working proteins.
 Transgenic plants: inserting genes to provide new
proteins, giving plants new properties
 DNA fingerprinting: analyzing pattern of bands that
are unique to an individual.
 Human Genome Project...
Biotech Tools
 The tools the scientists
use are very specific to
DNA and its
environment.
 The DNA first has to
be cut out of the
source organism
 The DNA has to be
isolated
 DNA can then be
introduced into host
DNA
Recombinant DNA
 Recombinant DNA is DNA from one source
organism being put into the DNA of a host organism.
1) Cutting Out DNA
 Restriction Endonucleases /
Enzymes are naturally occurring
enzymes that act like a pair of
molecular scissors to cut DNA
in a predictable and precise
manner, at a specific nucleotide
sequence called a recognition
site.
Discovery
 Hamilton Smith, John Hopkins University, won
the Nobel Prize in 1978 for discovering
restriction enzymes in bacteria.
 He found their main purpose was to cut foreign
DNA that tried to invade a bacterial cell (ie
DNA from a virus).
Naming System
 Restriction enzymes are named according to
the bacteria from which they originate.
 BamHI is from Bacillus amyloliquefaciens,
strain H. The I indicates it was the first
endonuclease isolated from that strain.
EcoRI - from Escherichia coli
BamHI - from Bacillus amyloliquefaciens
HindIII - from Haemophilus influenzae (the one H. Smith found)
PstI - from Providencia stuartii
Sau3AI - from Staphylococcus aureus
AvaI - from Anabaena variabilis
Recognition sites
 4 – 8 base pairs in length.
 Palindromic: both strands have the same
sequence when read in the 5' to 3' direction.
 Ex. HincII recognizes the following
sequences:
5'-G T C GA C-3'
3'-C A G C T G-5'
5'-G T T G A C-3'
3'-C A A C T G-5'
5'-G T C A A C-3'
3'-C A G T T G-5'
5'-G T T A A C-3'
3'-C A A T T G-5'
 The restriction enzyme EcoRI binds to 5'-GAATTC-3'
3'-CTTAAG-5'
 EcoRI breaks the phosphodiester bond between G
and A,
 then it pulls apart the two strands by breaking the Hbonds between the complementary base pairs.
 Produces what are called sticky ends (unpaired
nucleotides at each end).
Sticky vs. Blunt
 Other restriction enzymes
like AluI produce blunt
ends, or ends with no
overhang.
 Sticky ends are usually
more helpful to molecular
biologists as they can
easily be joined with
other DNA fragments cut
by the same restriction
enzyme.
 Blunt ends are harder to
fuse to a foreign DNA

p281 #1-5
molecule.
 A host must protect its own DNA from endonucleases.
 Methylases are enzymes that place a methyl group
(CH3) on recognition sites
 This prevents the restriction enzyme from cleaving the
DNA at that spot.
 Host DNA is methylated, but foreign DNA is not, so it
can be cut by the host cell's restriction enzymes.
2)Isolating DNA Fragments
 Scientists make use of restriction
endonucleases to cleave DNA into smaller
fragments
 Gel electrophoresis is used to isolate the
required gene segment from the rest of the
DNA
Gel Electrophoresis
 The fragments of DNA will
be run through a porous
agarose gel using
electricity.
 The fragments of DNA
are pulled through pores
in the gel due to their
negative charge.
 Smaller fragments will
move faster than larger
because they can fit
through the pores better.
http://www.stanford.edu/group/hopes/diagnsis/gentest/f_s02gelelect.gif
Steps:
 Solutions of fragments are placed in wells
(depressions at one end of the gel)
 The DNA is mixed with a dye so it will be seen as it
moves through the gel.
 Markers are usually put in the first well, These are
pieces of DNA whose size is known. They help
determine the length of the unknown DNA
fragments.
 The gel is submerged in a buffer solution and
connected to a power source.
 The anode will be at the top and the cathode at
the bottom. DNA is negatively charged, it will
move away from the anode to the cathode.
 The power source is only left on for a set amount
of time, so the fragments don’t move all to the
end or run off the gel, you want them separated
on the gel.
VIEWING THE GEL
http://www.mcps.k12.md.u
s/departments/intern/stp/im
ages/gel_electrophorsis.jp
g
http://www.life.uiuc.edu/molbio/geldigest/fullsize
/geldraw.jpg
 The gel is stained with ethidium bromide which
will cause the gel to fluoresce under UV light.
 The band of the DNA fragments can be seen and
the researcher is able to compare samples from
various sources or isolate a DNA fragment they
want to purify.
3) INTRODUCING FOREIGN
DNA INTO A HOST: PLASMIDS
and TRANSFORMATION
 Plasmids are
 small (1000 to 200
000bp in length),
 circular DNA
molecule
 independent of the
bacterial
chromosome.
 Plasmid DNA can be
replicated using the
bacterial cell’s
machinery.
http://www.rpgroup.caltech.edu/courses/PBL/images_dna
science/pZ%20Plasmid.gif
Plasmids
 Beneficial because they often contain important
genes such as antibiotic resistance, heavy metal
protection.
 Plasmids are used by biologists to incorporate genes
they want replicated or transcribed/translated in vast
amounts in little time into bacterial cells.
 Vector: vehicle used to introduce DNA into a host cell,
ie a plasmid or virus.
3) INTRODUCING FOREIGN DNA
INTO A HOST
 If we can cut genes out,
we must be able to join
them to foreign DNA.
 When sticky ends join
together, DNA ligase
recreates the
phosphodiester bonds.
 Blunt ends cannot be
joined by our own DNA
ligase, they must be
joined by
, an
enzyme from the T4
bacteriophage (virus).
STEPS:
1. Restriction enzymes
are used to cut out
the gene from the
original cell AND to
open the bacterial
plasmid.
2. Once the foreign gene
is isolated it can then
be inserted into the
plasmid. The plasmid
is now considered
recombinant DNA.
http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gif
TRANSFORMATION
3. The recombinant
DNA is then
introduced into a
bacterial cell.
Sometimes a host
cell must be
manipulated to take
up the foreign DNA
plasmid.
 Transformation: introduction of foreign DNA
(usually by plasmid or virus) into a bacterial cell.
 Host cell: cell that has taken up foreign plasmid or
virus and whose cellular machinery is being used to
express the foreign DNA.
 Competent cell: cell that readily takes up foreign
DNA.
4) Selection and Cloning
 Cells that have been successfully transformed
must be isolated (usually by antibiotic
resistance)
 The vectors used for cloning usually carry an
antibiotic-resistance gene. Growth of colonies
on media containing the antibiotic indicates
successful transformation.
Cloning
 Colonies are isolated from media and grown in
culture to produce multiple copies (clones) of
the recombinant DNA
 When the bacteria replicates the recombinant
DNA plasmid, the new gene product will be
formed multiple times (ie. the gene is cloned).
PCR – another means of
copying DNA in large numbers
 stands for Polymerase Chain Reaction,
 developed in the late 1980's by Kary Mullis; awarded
Nobel Prize in Chemistry in 1992.
 Does not require a plasmid. The fragment is copied
directly.
 Useful for forensic criminal investigations, medical
diagnosis, genetic research. Only small amounts of
DNA are needed.
PCR Process
 PCR is amplification of a DNA sequence by
repeated cycles of strand separation and
replication in the laboratory (DNA photocopying).
http://users.ugent.be/~avierstr/principles/pcrcopies.gi
Steps of PCR
1. Strands are separated using heat
2. DNA primers, synthesized in the lab, are
created to complement the start of the target
area to be copied.
3. Temp is decreased and the primers anneal
4. Taq polymerase (from bacteria) creates new
strands of target area
5. Sequence is repeated over and over on each
of the new strands built
Restriction Fragment Length
Polymorphism (RFLP)
 Entire genome is
subjected to restriction
enzyme digestion
 DNA run on an agarose
gel, using gel
electrophoresis
 Single stranded DNA
transferred to a
membrane
http://homepage.smc.edu/HGP/images/rflp.gi
f
RFLP
 ssDNA hybridized with
radioactive probes for
specific regions (such as
alleles or areas known as
variable number tandem
repeats, that lead to a
specific disease).
 An X-ray film is
developed, called an
autoradiogram, and the
pattern can then be used
to identify a suspect, or
detect a genetic mutation.
SEQUENCING DNA
 Sanger dideoxy method: uses DNA replication and
dideoxy nucleotides to determine the complementary
strand.
 Developed by Frederick Sanger and colleagues at
Cambridge University in Great Britain in 1977. They
used it to sequence the genome of a bacteriophage
(viral DNA) 5386 base pairs long.
Sanger dideoxy method
 Dideoxy nucleotides are
missing the -OH group
on carbon 3 and
therefore inhibit the
process of replication.
 Every time one is added,
the process stops and
only small sequences are
created.
 These sequences can
be run on a gel, and
since they will run from
shortest to longest, you
can actually read the
sequence by knowing
which dideoxy
nucleoside was used
and therefore stopped
replication at each point.
Fluorescent Detection of
Oligonucleotides
 The Human Genome
Project used a similar
method, but also included
fluorescence on each
dideoxy nucleoside, so
the A, G, T and C's lit up
as different colours.
 A computer reads the
sequence from gel
electrophoresis.
 Thousands of sequencers
worked 24 hours a day, 7
days a week to decipher 3
billion base pairs.