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Biotechnology
GENETIC MODIFICATIONS and BIOTECHNOLOGY
Genetic engineering:
the sequence of DNA
altering
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.
Techniques are now commonplace in molecular biology labs
worldwide.
Other examples:
Insulin, 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 AIDSassociated wasting syndrome now
BIOTECHNOLOGY
Biotechnology involves the manipulation of DNA and protein
synthesis.
Molecular biologists analyze and alter genes and their respective
proteins
Examples:
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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...
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 the host 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 .
Hamilton Smith, John Hopkins University, won the Nobel Prize
in 1978 for discovering restriction enzymes in bacteria (Hind III).
He found their main purpose was to cut foreign DNA that tried to
invade a bacterial cell (i.e. DNA from a virus).
Restriction enzymes are named according to the bacteria from
which they originate.
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Bam HI is from Bacillus amyloliquefaciens, strain H.
The I indicates it was the first endonuclease isolated
from that strain.
Recognition sites are usually:
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4 – 8 base pairs in
length.
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Palindromic: both
strands have the same
sequence when read
in the 5' to 3' direction.
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Table 1 p 279:
examples
The restriction enzyme EcoRI binds to 5'-GAATTC-3' / 3'-CTTAAG-5'
EcoRI finds this recognition site and breaks the phosphodiester bond
between G and A, then it pulls apart the two strands by breaking the
H-bonds between the complementary base pairs.
Produces what are called sticky ends (unpaired nucleotides at each
end).
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 molecule.
A host must protect its own DNA from endonucleases.
Methylases are enzymes that place a methyl group (CH3) on
recognition sites which 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: GEL ELECTROPHORESIS
Restriction endonucleases cleave the
DNA into smaller fragments
Gel electrophoresis is used to isolate the
required gene segment from the rest of the
DNA
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_s02
gelelect.gif
http://content.answers.com/main/content/wp/en/a/ab/Agarose_Gel_Electrophoresis.png
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 itself is made up of polyacrylamide or agarose.
Dye is added to the wells to help visualize the DNA segments.
Usually in the first well, DNA markers of set length are added, so the scientist
can estimate the sizes of the pieces in the other wells.
Once the gel has finished, it is stained using ethidium bromide, a carcinogen
that can attach to DNA and fluoresce under UV light.
Once the scientist finds the DNA piece he/she wants, they can excise it out of
the gel and purify it and use it for whatever purpose they need it for.
3) INTRODUCING FOREIGN DNA INTO A HOST: PLASMIDS and
TRANSFORMATION
http://www.rpgroup.caltech.edu/courses/PBL/images_dna
science/pZ%20Plasmid.gif
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.
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
1.
2.
3.
4.
Generation of DNA fragments using restriction endonucleases
Construction of a recombinant DNA molecule
Introduction into a host cell
Selection
Cells that have been successfully transformed must be
isolated (usually by antibiotic resistance)
The vectors used for cloning usually carry an antibioticresistance gene. Growth of colonies on media containing the
antibiotic indicates successful transformation.
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. Therefore when the process is
finished, it is not necessary to remove the plasmid from the
bacteria and the desired gene from the 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).
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
After about 30 cycles more than 1 billion copies of the targeted area
will exist (230).
http://users.ugent.be/~avierstr/principles/pcrcopies.gi
RFLP
“Restriction fragment length polymorphism”
Entire genome is subjected to restriction
enzyme digestion
DNA run on an agarose gel, using gel
electrophoresis
Single stranded DNA transferred to a membrane
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.
http://homepage.smc.edu/HGP/images/rflp.gi
f
SEQUENCING DNA
Sanger dideoxy method: uses DNA replication and dideoxy
nucleoside triphosphates 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 nucleosides are
missing the -OH group on
carbon 3 and therefore
inhibit the process of
replication.
- Everytime 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
read the sequence from gel
electrophoresis. Thousands
of sequencers worked 24
hours a day, 7 days a week to
decipher 3 billion base pairs.