Chapter 13:
DNA Technology
With all our knowledge of DNA and genes, is
there any way to manipulate DNA?
• Genetic Engineering –
form of applied genetics in which
genes/DNA are manipulated
Ex. Genetic engineers believe they can improve
the foods we eat. Tomatoes are sensitive to frost.
This shortens their growing season. Fish, on the
other hand, survive in very cold water. Scientists
identified a particular gene which enables a
flounder to resist cold and used the technology
of genetic engineering to insert this 'anti-freeze'
gene into a tomato.
DNA Technology
Science involved in the ability to manipulate
genes/DNA
Purpose:
1. Treat diseases (Cystic fibrosis)
2. Treat genetic disorders (hemophilia,
diabetes)
3. Improve food crops (better tasting veggies,
longer shelf life, fungus resistance)
4. Improve human life in general (vaccines…)
How is it all done?
Recombinant DNA Technology
Steps in the process:
-I. Isolation of DNA = extraction
- II. Copying DNA with PCR
-III. Cutting DNA with restriction
enzymes
-IV. Comparing DNA with gel
eletrophoreisis
OR – V. use as vectors in
recombinant DNA
I. Isolate DNA.
•
•
Remove tissue from organism
(You will do this in your DNA extraction
lab)
Store at 4°C
PCR – Polymerase Chain
Reaction
A genetic copy machine
• The polymerase
chain reaction (PCR)
is a rapid way of
amplifying
(duplicating)
specific DNA
sequences from a
small sample of
DNA.
II. Copy DNA - PCR
• Polymerase Chain Reaction
• Uses DNA sample, DNA polymerase
from T. aquaticus (hot springs)
• Think of this process as a molecular
copying machine
III. Cutting DNA - Restriction
enzymes
– Enzymes that can cut at
particular locations in the
DNA
- DNA engineering today is
totally dependent on
restriction enzymes
-Restriction enzymes are
endonucleases
- Think of them as molecular
scissors
What are restriction enzymes?
• Bacterial enzymes – used to cut DNA
Different bacterial strains express
different restriction enzymes
• The names of restriction enzymes are
derived from the name of the bacterial
strain they are isolated from
• Cut (hydrolyze) DNA into defined and
REPRODUCIBLE fragments
• Basic tools of gene cloning
Names of restriction endonucleases
• Titles of restriction enzymes are derived from
the first letter of the genus + the first two
letters of the species of organism from which
they were isolated.
• EcoRI - from Escherichia coli
• BamHI - from Bacillus amyloliquefaciens
• HindIII - from Haemophilus influenzae
• PstI - from Providencia stuartii
• Sau3AI - from Staphylococcus aureus
• AvaI - from Anabaena variabilis
Source microorganism
Enzyme
Arthrobacter luteus
Alu I
Bacillus amyloiquefaciens
H
Bam HI
Escherichia coli
Haemophilus gallinarum
Haemophilus infulenzae
Providencia stuartii 164
Eco RI
Hga I
Hind III
Pst I
Not I
Staphylococcus aureus 3A Sau 3A
Nocardia otitiscaviaruns
Serratia marcesans
Thermus aquaticus
Sma I
Taq I
Recognition Site
Ends
produced
AG CT
Blunt
G GATCC
Sticky
G AATTC
GACGC(N)5
Sticky
Sticky
A AGCTT
CTGCA G
Sticky
Sticky
GC GGCCGC Sticky
GATC
Sticky
CCC GGG
T CGA
Blunt
Sticky
Restriction enzymes recognize a specific
short nucleotide sequence
• For example, EcoRI recognizes the
sequence
•
5‘- G/ A A T T C -3'
•
3'- C T T A A /G -5'
Examples of restriction enzymes and the
sequences they cleave
•Palindromes – same base pairing forward and backwards
Let’s try some cutting:
•
•
•
Using this piece of DNA, cut it with Eco RI
• G/AATTC
GACCGAATTCAGTTAATTCGAATTC
CTGGCTTAAGTCAATTAAGCTTAAG
•
•
GACCG/AATTCAGTTAATTCG/AATTC
CTGGCTTAA/GTCAATTAAGCTTAA/G
What results is:
• GACCG AATTCAGTTAATTCG AATTC
• CTGGCTTAA GTCAATTAAGCTTAA G
Sticky end
Sticky end - tails of
DNA – easily bind
to other DNA
strands
Blunt & Sticky ends
• Sticky ends – Creates an overhang. BamH1
• Blunts- Enzymes that cut at precisely
opposite sites without overhangs. SmaI is
an example of an enzyme that generates
blunt ends
IV. Gel Electrophoreisis
• V. Analysis of DNA
DNA fingerprinting –
– Banding pattern of the fragments of cut
DNA on a special gel medium (agarose)
Purpose for DNA fingerprinting
• Comparing banding
patterns to determine
hereditary relationships
between people
• Comparing banding
patterns of 2 different
species to determine
evolutionary
relationship
• Compare samples of
blood or tissue for
forensic purposes (who
done it?)
•Very accurate method of accessing DNA – 99.99%
•Odds – 1 in 1,000,000,000
• Does not work with identical twins
•Use strands of DNA that have a lot of VNTRs
•
How is it done?
VNTR analysis – variable number tandem repeats - we
each have non-coding segments on our DNA. Fragment
lengths varies with each person
1. Extract DNA sample from blood or tissues
2. Cut DNA using restriction enzymes. Separate fragments by gel
electrophoresis – separates DNA fragments by the # of base pairs
(length of the fragment) and charge
3.
Place DNA sample into wells in the agarose gel – molecular sieve
4.
Run a current through the gel. The DNA (negatively charged) will
migrate from (-) to (+)5.The larger fragments will not migrate that far.
The small fragments will go the furthest
5.
Stain gel and bands in a dye or use a radioactive probe to analyze
the banding
Now that you have the desired gene or
piece of DNA, what do you do with it?
• V. Recombinant DNA
– Transfer of isolated gene to another
organism with the purpose of having the
organism transfer the gene to another.
– Use bacterial plasmids
1. The same restriction enzyme used to
cut the desired gene, is used to splice
the plasmid
2. Donor gene (desired gene) is then
spliced or annealed into the plasmid
3. Plasmid is then returned to bacterium
and reproduces with donor gene in it.
4. Bacterium with donor gene can
transfer donor genes to organisms it
infects.
How this works to help humans
• Looking at the gene that produces insulin for the
treatment of diabetes:
1. Isolate insulin gene from a healthy human
2. Using a restriction enzyme, cut out insulin
producing gene
3. Cut bacterial plasmids with same restriction
enzyme
4. Introduce human insulin producing gene to
bacterial plasmid
5. Bacterial plasmid takes up gene - recombinant
DNA
6. Bacterial plasmid reproduces and starts
expressing insulin produce gene
7. Insulin is produced and harvested from bacteria.
• What has been produced is Recombinant
DNA - DNA with genes from other
organisms
• Transgenic organisms have introduced
DNA from another species in them
Practical Use of DNA technology
•
•
•
Pharmaceutical products – insulin, HBCF
(human blood clotting factor)
Genetically engineered vaccines – to
combat viral infections (pathogenic –
disease causing) – your body recognizes
foreign proteins, produces antibodies.
Introduced viral proteins will trigger an
immune response and the production of
antibodies
Altering viral genomes – makes them no
longer pathogenic – now a vaccine
Increasing agricultural yields –
– New strains of plants – GMO – Genetically Modified
organism. Try this one!!
– Insect resistant plants – Insert gene that digests larvae
when larvae try to eat the plant – Not always specific to
harmful species!! – Monarch problem
– Disease resistance – Fungal resistance in tomatoes, corn,
soybean
– Herbicide resistance - *Round Up won’t harm the good
plants, only the bad plants (weeds) – cheaper and less
labor extensive than weeding
– Getting genes from Nitrogen fixing bacteria inserted into
plants – fix their own nitrogen (a must for plants) in N
poor soils
– Salt tolerant plants – can grow plants where high
concentrations of salt in the air or soil
• Improve quality of produce
- Slow down the ripening process –
ship when unripened, to market
when ripe
- Enhance color of produce
- Reduce hairs or fuzz on produce
- Increase flavor
Why GM Foods?
Safety and Environmental Issues
• All food products are regulated by the:
Food and Drug Administration – FDA
• Nat’l Institutes of Health Recombinant DNA
Advisory Committee and the Department of
Agriculture (USDA)
• Environmental Protection Agency (EPA)
• All set standards for safety procedures and
require permits and labeling (not in US
though). Look for a 8 before the product
code. 84011 – GMO banana
• Problem with transgenic foods is that an
introduced gene may produce a protein that
someone may be sensitive to. FDA does not
require that on a label
Gene Therapy
• Treatment of a genetic
disorder (like cystic fibrous)
by correcting a defective
gene that causes a
deficiency of an enzyme.
• Nasal spray that carries
normal enzyme gene. Body
makes enzyme and patient
breathes normally. Regular
treatments necessary
• Has not been proven to be
successful in the long term
CLONING
Hello Dolly!