Download Recombinant DNA

If your eyes follow the movement of the rotating pink dot, you will only see one color, pink. If you
stare at the black + in the center, the moving dot turns to green. Now, concentrate on the black + in
the center of the picture. After a short period of time, all the pink dots will slowly disappear, and
you will only see a green dot rotating if you're lucky! It's amazing how our brain works. There
really is no green dot, and the pink ones really don't disappear. This should be proof enough, we
don't always see what we think we see.
Recombinant DNA
Microbiology 2314
California 2003
• In Sacramento, a group of
scientists have genetically
altered fish. Basically they
took ordinary zebra fish,
added genetic jellyfish stuff,
and made pet fish that glow
in the dark, when placed
under a blacklight. They hit
the shelves in 2004 to be
sold as pets.
• They will be on sale, just about everywhere except
California (which bans lab-engineered species.)
So…We can genetically engineer
all the animals we want, just can’t
sell em.
Note:
• The genetic material of each living
organism-plant or animal, bacterium or
virus-possesses sequences of its nucleotide
building blocks (usually DNA, sometimes
RNA) that are uniquely and specifically
present only in its own species.
What is Genetic Engineering?
• Genetic engineering (GE) is the transfer of genes
from one organism to another through means that
do not occur in nature, but through human
intervention. This involves isolating and then
moving genes within and without different species
by recombinant DNA techniques and other
manipulation of the genetic construct outside the
traditional practices such as sexual and asexual
breeding, hybridization, fermentation, in-vitro
fertilization and tissue culture.
Introduction
• Biotechnology
- The use of microorganism cells or cell
components to make a new product.
• Idea More than 60 Years Old
• Delay Due to Technology
1. Cut
2. Combine
3. Reinsert
Advent of Recombinant DNA
• Closely Related Organisms Can Exchange
Genes
• Labs Can Facilitate Transfer Between
Unrelated Species
• DNA Transfers Genes
• Recombinant DNA Technology is a term
used to refer to experimental protocols
leading to the transfer of genetic
information (DNA) from one organism to
another.
Tools and Techniques
•
•
•
•
Restriction Enzyme Makes Cuts
Cuts at Specific DNA Sequences
Accurate
75 Known Restriction Enzymes
• Restriction enzymes take apart the DNA in
a certain area and allow for a plasmid to be
inserted within the gap that is created.
Sticky Ends/Blunt Ends
DNA Ligase
• Vectors
- Called Chimeras
• Shuttle Vectors
- Plasmids that can exist in several
different species.
• Transformation
- Process by which a new gene is
inserted into a cell
Process
• Isolate the Source and Vector DNA
• Use Restrictive Enzymes to Make Cuts in
Both
• Mix Plasmid and Vector DNA Together and
Bond Via Ligase
• Clone
Overview of Process
Properties of a Good Vector
1. It should be able to replicate
autonomously. When the objective of
cloning is to obtain a large number of
copies of the DNA insert, the vector
replication must be under relaxed
control so that it can generate multiple
copies of itself in a single host cell.
2. It should be easy to isolate and purify.
3. It should be easily introduced into the host
cells. In other words transformation of the
host with the vector should be easy.
4. The vector should have suitable marker genes
that allow easy detection and/or selection of
the transformed host cells.
5. When the objective is gene transfer, it should
have the ability to integrate either itself or
the DNA insert it carries into the genome of
the host cell.
6. The cells transformed with the vector
containing the DNA insert (recombinant
DNA) should be identifiable be selectable
from those transformed by the unaltered
vector.
7. A vector should contain unique target
sites for as may restriction enzymes as
possible into which the DNA insert can
be integrated.
8. When expression of the DNA insert is
desired, the vector should contain at
least suitable control elements, e.g.,
promoter, operator and ribosome
binding sites.
Plasmids Serve As Vectors
• Plasmids are considered "replicons",
capable of autonomous replication within a
suitable host.
• Plasmids can be found in all three major
domains: Archea, Bacteria and Eukarya.
• Similar to viruses, plasmids are not
considered by some to be a form of "life".
Methods for Inserting Foreign
DNA Into Cells
• Cells can take up naked DNA by
transformation. Chemical treatments are
used to make cells that are not naturally
competent take up DNA.
• Pores (holes) can be made in protoplasts
and animal cells by electric current during
the process of electroporation to provide an
entrance for DNA.
• Foreign DNA can be introduced into plant
cells by shooting DNA-coated articles into
the cells.
• Foreign DNA can be injected into animal
cells by using a fine glass micropipette.
Why Genetic Engineering?
• 3000 Known Genetic Diseases
Crop Improvement
• The improvement of crops
with the use of genetics
has been occurring for
years. Traditionally, crop
improvement was
accomplished by selecting
the best looking
plants/seeds and saving
them to plant for the next
year’s crop.
DNA Extraction
• DNA extraction is the first
step in the genetic
engineering process. In
order to work with DNA,
scientists must extract it
from the desired organism.
A sample of an organism
containing the gene of
interest is taken through a
series of steps to remove
the DNA.
Gene Cloning
• The second step of the
genetic engineering
process is gene cloning.
During DNA extraction,
all of the DNA from the
organism is extracted at
once. Scientists use gene
cloning to separate the
single gene of interest
from the rest of the genes
extracted and make
thousands of copies of it.
Gene Design
• Once a gene has been cloned, genetic engineers
begin the third step, designing the gene to work
once inside a different organism. This is done in a
test tube by cutting the gene apart with enzymes and
replacing gene regions that have been separated.
Transformation
• The modified gene is now
ready for the fourth step in
the process, transformation
or gene insertion.
• Since plants have millions of
cells, it would be impossible
to insert a copy of the
transgene into every cell.
Therefore, tissue culture is
used to propagate masses of
undifferentiated plant cells
called callus. These are the
cells to which the new
transgene will be added.
Backcross Breeding
• The fifth and final part of
producing a genetically engineered
crop is backcross breeding.
Transgenic plants are crossed with
elite breeding lines using
traditional plant breeding methods
to combine the desired traits of
elite parents and the transgene into
a single line. The offspring are
repeatedly crossed back to the elite
line to obtain a high yielding
transgenic line. The result will be
a plant with a yield potential close
to current hybrids that expresses
the trait encoded by the new
transgene.
Common Terms (FYI Only)
• Agbiotech = the agricultural arm of the biotechnology
industry
• Biotech = the biotechnology industry
• GE = genetic engineering/genetically engineered
• GM = genetically modified
• GMO = genetically modified organism
• Pharm crop = a GE crop that creates its own
pharmaceutical byproducts in virtually all parts of the
plant
• Transgenic = another name for GE
GMO / Genetically Modified
Organism
• A GMO is a plant, animal or microorganism (e.g.,
bacteria) that is created by means that overcome
natural boundaries.
• Genetic engineering involves crossing species
which could not cross in nature.
• For example, fish genes have been inserted into
strawberries.
• The most widely grown GE crops are soybeans,
corn, canola (rapeseed) and cotton. Nearly all GE
crops grown today are one of two varieties: "insect
resistant" and "herbicide tolerant" crops.
Good Reasons Do Exist
• For example, the splicing of a specific
flounder gene for producing a unique blood
"antifreeze" protein into tomatoes, to render
them frost resistant; splicing insect proteins
into zucchinis to create a taste and fragrance
that is repugnant to other insect pests; or
growing potatoes endowed with built-in
pesticides.
According to the FDA
• While the Food and Drug Administration
insists that foods produced by genetic
engineering are the same as foods from
traditional breeding, their own scientists
reported that, "the processes of genetic
engineering and traditional breeding are
different and... they lead to different risks."
GENETIC ENGINEERING: A
CAUTIONARY APPROACH
• The Institute of Science,
Technology and Public Policy has
taken a strong precautionary stand
on genetic engineering. In
collaboration with leading
scientists and other public service
organizations, it has launched a
nation-wide public awareness
campaign to alert the public about
the dangers of genetically
engineered foods, and is calling
for rigorous safety testing and
mandatory labeling of such foods.
Humans?
• The biotech industry's rationale for the
genetic engineering of humans is the
predisposition of human beings to certain
diseases. If such human frailties could be
fortified by genetically transplanting traits
of other animals, insects, bacteria or viruses,
then it might be possible for
biotechnologists to improve upon our
species.
The Argument
• Genetic surgery performed on fetuses would, with
high probability, infect the germ line (egg or
sperm) cells.
• As a consequence, any such genetic defects would
be passed on to future generations, causing
irreversible gene pollution and the potential for
new genetic diseases.
• In addition to the immediate and long-term gross
health risks posed by irreversible gene pollution,
we have no idea what the subtle effects of
incomplete or mutated human DNA will be on the
human race.
The Fact Exists That There Are Some Diseases Out
There We Cannot Cure Without Genetic
Engineering.
Take a Stand. Have a View.
But Ask Yourself…Would That View Change If It Were
Your Child?
Real Life
Example
SCID
Babies born without immune systems
quickly become ill during weaning when
the protection of their mother's milk –
which contains maternal antibodies –
begins to wear off. Without
transplant medicine of some kind, the
only way of keeping these children alive
is to cocoon them in a sterile
environment free of potentially lethal
microbes.
In the Sixties and Seventies, hospitals
put babies with severe combined
immune deficiency (SCID) in plastic
"bubbles" where the the air is filtered
and direct contact with the outside
world is minimized.
Paul Simon
Boy in the Bubble
Rhys Evans (April 2002 UK)
• Gene Therapy used to
cure a toddler of
SCID
• Took Bone Marrow
from the child, then
used a virus to carry a
new version of the
gene into the immune
cells from the
marrow.
PCR
• What is a polymerase? A polymerase is a
naturally occurring enzyme, a biological
macromolecule that catalyzes the formation and
repair of DNA (and RNA).
• The accurate replication of all living matter
depends on this activity -- an activity scientists
have learned to manipulate.
• In the 1980s, Kary Mullis at Cetus Corporation
conceived of a way to start and stop a
polymerase's action at specific points along a
single strand of DNA.
PCR
• The polymerase chain reaction (PCR) is a
scientific technique in molecular biology to
amplify a single or a few copies of a piece of
DNA across several orders of magnitude,
generating thousands to millions of copies of a
particular DNA sequence.
A strip of eight PCR
tubes, each containing
a 100 μl reaction
mixture
• The purpose of a PCR (Polymerase Chain
Reaction) is to make a huge number of
copies of a gene. This is necessary to have
enough starting template for sequencing.
• There are three major steps in a PCR, which
are repeated for 30 or 40 cycles. This is
done on an automated cycler, which can
heat and cool the tubes with the reaction
mixture in a very short time.
3 Major Steps to PCR
1. Denaturation at 94°C :
2. Annealing at 54°C :
3. Extension at 72°C :
1. Denaturation at 94°C :
During the denaturation, the double strand melts open
to single stranded DNA, all enzymatic reactions stop
(for example : the extension from a previous cycle).
2. Annealing at 54°C :
The primers are jiggling around, caused by Brownian
motion. Ionic bonds are constantly formed and broken
between the single stranded primer and the single
stranded template. The more stable bonds last a little
bit longer (primers that fit exactly) and on that little
piece of double stranded DNA (template and primer),
the polymerase can attach and starts copying the
template. Once there are a few bases built in, the ionic
bond is so strong between the template and the primer,
that it does not break anymore.
3. Extension at 72°C :
This is the ideal working temperature for the polymerase.
The primers, where there are a few bases built in, already
have a stronger ionic attraction to the template than the
forces breaking these attractions.
Primers that are on positions with no exact match, get
loose again (because of the higher temperature) and don't
give an extension of the fragment.
The bases (complementary to the template) are coupled
to the primer
Why PCR?
• PCR, the quick, easy method for generating
unlimited copies of any fragment of DNA,
is one of those scientific developments that
actually deserves timeworn superlatives like
"revolutionary" and "breakthrough."
To Sum It All Up
• PCR makes it possible to have enough DNA
to trace genetic material back to its origin,
identifying with precision at least what
species of organism it came from, and often
which particular member of that species.
Gene Cloning
• Gene cloning is the process in which a gene of
interest is located and copied (cloned) out of
DNA extracted from an organism.
• When DNA is extracted from an organism, all of
its genes are extracted at one time. This DNA,
which contains thousands of different genes. The
genetic engineer must find the one specific gene
that encodes the specific protein of interest.
• Since there is no way to locate a gene by
visibly looking at all of the DNA, scientists
must use other methods such as …
1. DNA Probes
2. Antibiotic-Resistance Genes
3. Gene Products
1. A DNA probe is a labeled segment of DNA
or RNA used to find a specific sequence of
nucleotides in a DNA molecule. Probes may
be synthesized in the laboratory, with a
sequence complementary to the target DNA
sequence.
If the DNA probe finds a match, it will adhere
to the target gene.
2. A very common method for identifying a
particular protein is by using antibodies, or
immunoglobulins, that bind specifically to
that protein. This is often done to select
antibiotic resistant genes.
3. A gene product is the biochemical material,
either RNA or protein, resulting from
expression of a gene. A measurement of the
amount of gene product is sometimes used to
infer how active a gene is.