Biotechnology
Biology III
Bellringer 1/10/12
• What are the 4 bases in DNA?
• What is Transcription?
• What is Translation?
What is Biotechnology?
• Biotechnology: is a field of applied biology
that involves the use of living organisms
and bioprocesses in engineering,
technology, medicine and other fields of
requiring bio products.
Selective Breeding
• Choose organisms with the desired traits
and breed them, so the next generation also
has those traits
– Nearly all domesticated animals and crops
– Luther Burbank (1849-1926) developed >800
diff varieties of plants in his lifetime
Selective Breeding
• For a long time, humans have selected the
best plants and animals to
breed
• Why?
• Examples?
• Milk Cows
– 1947 - produced 4,997 lbs... of milk/year
– 1997 - produced 16,915 lbs.... of milk/year
• Increasing the frequency of desired alleles
in a population is the essence of genetic
technology
Hybridization
• The act or process of mating organisms of different varieties or
species to create a hybrid.
• In plants – often results in better lines – hybrids are larger, stronger,
etc
• In animals – hybrids produced may be weaker and sterile
– Ex – wolf x dog ---- weak wolf-dog
– Ex – horse x donkey ---- mule (sterile)
Inbreeding
• Breeding two organisms that are
very similar to produce offspring
with the desired traits.
– Ex – dog breeds
•Risks – might bring together two
individuals that carry bad recessive genes –
many purebred dogs have genetic disorders
that mutts don’t get.
Inbreeding
• Mating between closely
related individuals
• Why?
• Done to make sure that
breeds consistently exhibit a
trait and to eliminate
undesired trait
– Creates purebred lines
• Can be bad also
– Can bring out harmful,
recessive alleles in a “family”
Increasing Variation
• Induce mutations – the ultimate source of
genetic variations among a group of
organisms
– Mutagens used – radiation and chemicals
– Some organisms are formed that have more
desirable variations.
Producing new kinds of bacteria
• Can expose millions of bacteria at one time
to radiation – increases chances of
producing a successful mutant.
– Ex – bacteria that can digest oil have been
produced this way
Producing new kinds of plants:
• Drugs that prevent chromosomal separation
in meiosis have been used to create plants
that have more than two sets of
chromosomes (2n). These are called
polyploid plants.
– Ex – bananas, citrus fruit, strawberries, many
ornamental flowers
Diploid corn
Tetraploid corn
What is Genetic Engineering?
• Making changes in the genetic code of a
living organism.
•
- Transferring of DNA/genes
from one organism to another
Also called recombinant DNA
technology or gene splicing.
-
What is Genetic Engineering?
• Genetic engineering can take place:
– Within a species (switching genes between
humans)
– Or between species (switching genes between
humans and bacteria)
• Why is this possible?
What is Genetic Engineering?
• Gene: holds the genetic information to
build and maintain an organism’s cells and
pass genetic traits to offspring.
What is Genetic Engineering?
• Genome: the entirety of an organism’s
heredity information.
How does genetic
engineering take place?
Manipulating DNA – tools of
the molecular biologist
1. DNA extraction – open the cells and
separate DNA from all the other cell parts.
Steps to DNA Extraction
1. Break the cells open to expose DNA
2. Remove membrane lipids by adding detergent
3. Precipitate DNA with an alcohol — usually
ethanol or isopropanol.
– Since DNA is insoluble in these alcohols, it will
aggregate together, giving a pellet upon
centrifugation. This step also removes alcoholsoluble salt.
2. Cutting DNA
• Sequences of DNA are isolated
using restriction enzymes.
• Use restriction enzymes
– each one cuts DNA at a
specific sequence of nucleotides.
(Usually 4-6 nucleotides)
• This will make different
lengths of DNA
What is the role/function of
restriction enzymes in bacteria?
Restriction Enzymes
• Many enzyme cut in palindromes
– Ex: a protein only cuts at AATT, it will cut the two
fragments at different points - not across from each
other (called sticky ends)
• Called sticky ends because they want to bond with things
due to their “open” end
Restriction Enzymes
• These sticky ends are beneficial, because if the
same enzyme is used in both organisms, they will
have identical ends and will bond with each other.
Restriction Enzymes
• The cut ends (because they are complementary)
can reattach or pair up with any other DNA
fragment or gene cut by the same restriction
enzyme.
•
http://www.youtube.com/watch?v=8rXizmLjegI
Restriction Enzymes
• Restriction enzymes are used to cut or cleave the
source DNA into fragments called: RFLP’s
(Restriction Fragments Length Polymorphism)
– Because the restriction enzyme’s recognition sequence
is likely to occur many times within the source DNA,
cutting will produce many fragments of different
lengths.
– Different RFLP’s may be made by using different
restriction enzymes that recognize different DNA
sequences.
Some Commonly Used
Restriction Enzymes
Eco RI 5'-G | AATTC
Eco RV 5'-GAT | ATC
Hin D III 5'-A | AGCTT
Sac I 5'-GAGCT | C
Sma I 5'-CCC | GGG
Xma I 5'-C | CCGGG
Bam HI I 5'-G | GATCC
Pst I I 5'-CTGCA | G
Plasmids
• Plasmids: is a DNA molecule that is
separate from and can replicate
independently of the chromosomal DNA
Examples of the insertion of
genes
Bellringer 1/19/11
• What does restriction enzymes do?
• How are restriction enzymes important for
genetic engineering?
• How has biotechnology affected you today?
– (Give at least one example)
3. Separating DNA
• RFLP can be separated (Based on their size)
by electrophoresis
– Since a 3-billion base sequence of the 4 DNA
nucleotides can produce more varied
combinations than there are humans, each of us
should have a unique DNA sequence.
Separating DNA – Gel Electrophoresis
1. Place fragments at one end of a porous gel –
we use agarose gel
2. Apply an electric current – The DNA is
negatively charged and will travel toward the
positive end of the gel.
3. The larger pieces of DNA move slower, the
smaller ones faster.
4. Used to compare genomes of different
organisms or different individuals.
5. Also used to locate and identify one particular
gene out of an individual’s genome.
Gel Electrophoresis
gslc
Click here for animation about
gel electrophoresis
Using the DNA Sequence
• Sequence can be read, studied, and changed.
• Techniques used to study DNA sequences:
– Use DNA polymerase and the 4 DNA bases to
produce a new DNA strand complementary to
unknown strand – some of the bases are dyed.
• Dye-labeled strands are then separated using gel
electrophoresis and the order of the bands tells the
DNA sequence of the unknown strand.
3. Recombinant DNA
• Once the DNA of interest is isolated it is
recombined with another organisms’ DNA
• Cell Transformation: A cell takes in DNA
from outside the cell.
– The external DNA becomes a component of
the cell’s DNA
Bacteria Transformation using
Recombinant DNA
• Cut a gene with a restriction enzyme out of a
human cell (ex – gene for insulin or growth
hormone work well)
• Cut a bacterial plasmid using the same restriction
enzyme (DNA ends will be complementary)
• Insert Human gene into bacterial plasmid
• Insert plasmid back into bacterial cell
• Bacteria will multiply, and all offspring will have
that gene – these bacteria will then follow the
directions of the human gene and make the protein
coded for (insulin or human growth hormone)
Transforming Cells
• Use bacterial plasmid to insert desired gene
into DNA
• Foreign DNA is first joined to a small, circular
DNA molecule known as a plasmid.
• Plasmids are found naturally in some bacteria and
have been very useful for DNA transfer.
Transforming Animal Cells
– Directly inject DNA into the nucleus of an egg
– it will become part of the chromosomes.
• Has been used to replace specific genes.
Glowing mouse cells in
embryos that were made
from sperm given the gene
for bioluminescence from
jellyfish – now all the cells
glow!
Making Recombinant DNA
• Step 1: To “recombine” or insert genes form
one organism
– Must first cut out the desired gene using the
restriction enzyme
– With the same restriction enzyme, cut out a
segment of DNA from a plasmid or virus.
Making Recombinant DNA
• Step 2: Because the two different sources of
DNA (human and bacteria) were cut with
the same restriction enzyme, the “sticky
ends will allow their DNA to recombine.
Making Recombinant DNA
• Step 3: Insert Human gene into bacterial
plasmid
– Insert plasmid back into bacterial cell
Making Recombinant DNA
• Step 4: Bacteria will
multiply, and all
offspring will have that
gene
– these bacteria will then
follow the directions of
the human gene and make
the protein coded
Checking for recombinant cells
• Recombinant molecules must be separated
from molecules consisting of just donor
DNA or plasmid DNA.
•
The experimenter designs the process so
that the plasmid contains two genes that
each enable a cell to grow in the presence of
a different antibiotic drug.
Applications of Genetic
Engineering
• Gene for luciferase was isolated from
fireflies and inserted into tobacco plants –
they glowed!
• Transgenic organisms – contain genes from
other species
A transgenic mouse,
which carries
a jellyfish gene,
glows green under
fluorescent light.
Transgenic Organisms
• Bacteria - Make human proteins like insulin
• Plants – 52% of soybeans, 25% of corn in
US in year 2000. Some produce natural
insecticide, some resist weed-killers, may
soon be used to produce human antibodies;
rice with vitamin A.
•Animals – mice
with immune
systems like
humans; farm
animals that grow
faster and larger
with extra copies of
growth hormone
genes; animals with
leaner meat;
chickens resistant to
bacterial infections.
Cloning
• Clone – member of a population
of genetically identical cells
produced from a single cell.
• 1996 – Dolly cloned –
1st mammal (sheep) cloned.
• She got arthritis several years
earlier than most sheep
• Died in 2003