recombinant DNA - Cloudfront.net

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Recombinant DNA
•recombinant DNA – techniques in which genes
from two different sources - often different species
- are combined in vitro into the same molecule
•This works because the genetic code is universal
•genetic engineering – the direct manipulation of
genes for practical purposes
•DNA technology has resulted 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
• genetic engineering is possible because of
restriction enzymes (restriction endonucleases):
• Very specific – recognize and then cut DNA
molecules at specific base sequences called a
restriction site (recognition sequence)
– These 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’.
• In nature, bacteria use restriction enzymes for
protection to cut foreign DNA (from invading
viruses)
• Restriction enzymes cut
the covalent bonds of both
strands, often in a
staggered way creating
single-stranded sticky
ends.
– Sticky ends will form
hydrogen-bonded base pairs
with complementary sticky
ends on other DNA
molecules cut with the same
restriction enzyme.
• DNA ligase bonds the
complementary sticky ends
together
• Restriction enzymes and
DNA ligase are used to “cut
and paste” DNA pieces
together
Bacterial Transformation
•Bacterial Transformation – scientists put
new genes into bacteria to develop
organisms that are beneficial to people –
uses include:
–Bacteria that can produce hormones
such as human growth hormone and
insulin
–Bacteria that eat oil slicks
Escherichia coli
• Often used for genetic engineering
• Common inhabitant of human colon – easy to
get
• Can be easily grown in suspension culture in a
nutrient such as Luria broth
• Has a simple circular chromosome with about
1/600th the haploid amount of DNA in a human
cell
• E. coli often contain small circular DNA
molecules called plasmids (extrachromosomal)
– confer a particular trait such as resistance to
antibiotics
– So we can easily introduce our own plasmids to
produce desired products
– Plasmids are produced by cutting desired DNA
(using restriction enzymes) and inserting a
gene into a plasmid to act as a carrier
– The gene is often inserted into a plasmid with
genes for antibiotic resistance so that the
transformed bacteria can be easily selected
from other cells that did not pick up the plasmid
• In nature, genes can be transferred
between bacteria in three ways:
– Conjugation – mating process during which
genetic material is transferred from one
bacterium to another of a different mating type
– Transduction – a virus acts as a vector (carrier)
to transfer small pieces of DNA from one
bacterium to another
– Bacterial Transformation – involves the transfer
of genetic information into a cell by direct
uptake of the DNA (occurs only rarely in
nature)
Transformation in the Laboratory
• Transformation was first performed in the
laboratory by Griffith and later by Avery,
MacLeod, and McCarty (experiment using
mice and pneumococcus bacteria)
• Bacteria can take up DNA only during the
period a the end of logarithmic growth – cells
are said to be competent (can accept DNA
that is introduced from another source)
• E. coli competence can be induced under carefully
controlled chemical growth conditions
• Plasmids can transfer genes and act as carriers for
introducing DNA from other bacteria or from
eukaryotic cells
• E. coli cell membrane is weakened using ice cold
CaCL2
• E. coli cells are then “heat shocked” to induce them
to take up the plasmid
• Sterile technique must be used
• Transformation Lab – we will transform bacteria by
introducing a plasmid that will convey resistance to
the antibiotic, ampicillin
• Ampicillin kills bacteria by interfering with their
ability to make cell walls
DNA Profiling
• Restriction Enzymes are also used for DNA
profiling
– Creating a pattern of DNA bands on a gel
• Because the restriction site (recognition sequence)
usually occurs (by chance) many times on a long
DNA molecule, a restriction enzyme will make
many cuts
• Result: production of fragments of DNA of various
lengths – Restriction Fragment Length
Polymorphs (RFLPs)
• Since all individuals have unique sequences of
DNA, restriction enzymes cut each individual’s
DNA into different sized RFLPs
• The RFLPs are then separated by gel
electrophoresis resulting in a bar-like pattern
• Electrophoresis means “to carry with an electric
current”
• Different sized RFLPs will be carried different
distances by an electric current as they migrate
through an agarose gel inside a gel box
– Electricity is run through the gel box creating a
positive end and a negative end
• Negatively charged DNA migrates from the
negative end of the gel box through the pores in
the gel to the positive end of the gel box
• Smaller RFLPs will migrate farther than larger
pieces, spreading the RFLPs across the gel in a
bar-like pattern
• Stain is used to make the DNA bands visible
SEM photo of a 1% LE Agarose gel at 22kX magnification
Uses for DNA profiling
• Allows scientists to compare DNA from
various organisms and identify a particular
individual (DNA can be extracted from
blood, saliva, hair roots, and skin)
• Crimework: rape and murder cases
(forensics)
• Paternity suits
• Missing persons and unidentified bodies
• Immigration disputes
• Animal work - breeding
Agricultural uses of DNA technology
Animal Husbandry – many farm animals are
treated with products made by recombinant
DNA methods (examples include vaccines,
antibodies, and growth hormones)
– some milk cows are injected with bovine
growth hormone (BGH) made by E. coli, in
order to raise milk production
– BGH also improves weight gain in beef
cattle
Transgenic animals – animals that contain genes from
another species have been developed for agricultural use
(examples include beef and dairy cattle, hogs, sheep and
several species of commercially raised fishes)
– modified DNA can be introduced into diary cows so that
they produce human proteins – protein is produced in
the milk – examples of medically important proteins that
have been produced in transgenic mammals include:
• blood clotting Factor VIII to treat hemophilia
• alpha-1- antitrypsin which helps protect the lungs
from damage during infections
– rainbow trout and salmon that are given a foreign
growth hormone can reach in one year a size that
usually requires 2 to 3 years of growth
Genetic engineering in plants
– plants have been genetically altered to
receive herbicide resistance (several
strains of cotton) – allows them to be
resistant to herbicides used to kill weeds
– some crop plants are being engineered to
resist infectious pathogens and pest
insects – reduces need to apply chemical
insecticides
• first genetically engineered fruits approved
by the FDA for human consumption were
tomatoes engineered with antisense genes
that retard spoilage
– researchers isolated gene responsible for
ripening
– they prepared a gene who's template strand had
a base sequence complementary to the normal
gene – an antisense version of the gene
– when spliced into the DNA of a tomato plant, the
antisense gene is transcribed into RNA that is
complementary to the ripening gene’s mRNA
– the antisense RNA binds to the normal mRNA,
blocking the synthesis of the enzyme causing
ripening and spoilage
Benefits and Possible Harmful
Effects of Genetic Modification
• See handouts and Clegg pg. 127-128
Cloning
• Clone – a group of cells, organisms, or
genes that are exact copies of each other
– Gene cloning – replication of donor genes in
bacterial or other host cells
– donor gene inserted into a bacterium is copied
every time the plasmid containing it replicates –
genes can be cloned by growing genetically
engineered bacteria
Polymerase Chain Reaction
• cloning a gene through genetic engineering can be timeconsuming and requires an adequate DNA sample as
starting material
• PCR technique allows researchers to amplify a tiny sample
of DNA millions of times in a few hours
• DNA polymerase uses nucleotides and primers to replicate
a DNA sequence in vitro, thereby producing two molecules
• Two strands of each molecule are then separated by
heating and replicated again, so then there are four,
double-stranded molecules
• After the next cycle of heating and replication there are
eight molecules, and so on
• Number of molecules doubles with each cycle
• PCR is useful in amplifying tiny samples of DNA ranging
from crime scenes to archaeological remains
•
•
Cloning organisms – cloning sometimes
occurs naturally (twins, asexual
reproduction)
organisms can be cloned artificially
(sheep, rabbits, toads and other sexually
reproducing animals have been cloned by
dividing up an embryo and transplanting
them into surrogate mothers)
Cloning of Dolly
1. sheep cloned from a non-reproductive cell
2. cell taken from udder of donor adult and cultured
in lab for 6 days
3. unfertilized egg taken from another sheep –
nucleus removed
4. egg without nucleus is fused with donor cell using
a spark of electricity
5. embryo resulting from fusion of udder cell and egg
transferred into the uterus of a third sheep who
acts as the surrogate mother
6. surrogate mother gives birth to lamb – lamb is
genetically identical to sheep that donated udder
cell
Stem Cells
• Cells that retain their ability to divide and
differentiate into various cell types
• Plants contain stem cells in their meristems
(reason why a cutting can grow into a new
plant)
• Embryonic stem cells are pluripotent – can
form any type of cell in an organism or can
form a complete organism
• Adult stem cells can divide to form new
body tissue cells (i.e. blood stem cells)
Therapeutic Uses of Stem Cells
• Embryonic stem cells are the most flexible
and can grow into any type of mature cell
– Parkinson’s and Alzheimer’s Disease can be
potentially treated by implanting stem cells that
could replace the damaged cells
Ethical Issues surrounding
therapeutic cloning
•
•
•
Therapeutic Cloning is the creation of an embryo to
supply embryonic stem cells for medical use
– Raises issue of whether it is right or wrong to
generate a new human embryo for medical
research
Two distinct forms of cloning:
1. Reproductive cloning – making copies of entire
organism
2. Therapeutic cloning – making copies of embryonic
stem cells
Opinions vary about whether both forms are
right/wrong or if one or the other is acceptable
Human Genome Project
•
•
A project that involved mapping the entire human
genome – determined the order of all the bases in
human DNA
Outcomes of the HGP:
1. Determine how many individual genes we have and
how they work.
2. Locating and determining the cause of genetic
disorders.
3. Development of gene therapies to treat genetic
disorders.
4. Comparing genetic makeup of human populations to
determine ancestries and how humans have migrated
and mixed their genes with other populations over
time.