Download DNA Technology Power Point 2016

DNA TECHNOLOGY
DNA Technology – science involved in the ability to
manipulate genes/DNA
Purpose:
 Cure disease (Cystic Fibrosis)
 Treat genetic disorders (Hemophilia, diabetes)
 Improve food crops (better tasting, longer shelf life, fungus
resistance…)
 Improve human life in general
 Helps us ID genes for traits
I. How could you get a desired trait without directly
manipulating the organisms’ DNA?
A. Selective Breeding
- choosing organisms with desired traits to
produce the next generation
Breeding the winners of a horse race (Smarty Jones)
Taking the seeds from the
Great Pumpkin
B. HYBRIDIZATION
Crossing organisms with different traits to produce a
hardier product
Ex. A mule is a cross of a horse and a donkey –
Sturdy and surefooted
Hybrid corn – tastes good and is more resistant to
disease.
C. INBREEDING
Maintaining the present genes by breeding only within the
population
Ex. Pedigree animals
Risk with dipping into the same gene pool and recessive traits
showing up that may be lethal or harmful.
D. INDUCING MUTATIONS
By using known mutagens, attempt to force mutations to
occur
Radiation & Chemicals
Not a sure bet nor do you know what you are going to get
Polyploidy (3N or 4N) plants have resulted from this – larger &
hardier
II. Manipulating Genes by Altering an Organisms
DNA
DNA Technology Purpose
Cure Diseases
Treat Genetic Disorder
Improve Food Crop
Improve Human Life (reproduce desired traits)
III. Practical Uses of DNA Technology (positive)
Pharmacutical Products
Genetically Engineered Vaccine
Increasing Agricultural Yields
(negative)
Allergies
GMO (genetically Modified Organisms)
Supperweeds
Cloning
Growing a population of genetically identical cells from a
single cell
Let’s discuss the positives and negatives of m cloning…..
Let’s do a little research first……
Lab Bio: Read Pros and Cons of Cloning
Honors Bio: Read The Real Face of Cloning
DNA TECHNOLOGY:
EX: 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
How do we copy a piece of DNA…..
The Tools:
DNA Extraction – Chemical procedure (we’ll do this)
Restriction enzymes – molecular scissors that cut DNA
at specific nucleotide sequences
Gel Electrophoresis – method to analyze fragments of
DNA cut by restriction enzymes through a gel made
of agarose (molecular sieve)
DNA Ligase – molecular glue that puts pieces of DNA
together
Polymerase Chain Reaction (PCR)- molecular copy
machine. Makes millions of copies of DNA/hr
Let’s suppose that you are a diabetic and can not
make your own insulin. What are you to do?
Inject insulin of course but from what source?
Old method was to use sheep insulin. Costly and labor
intensive
New method: Let bacteria with a human insulin producing
gene make it for you
THE METHOD:
Transformation of a bacterium to produce
human insulin
1. Extract the insulin producing gene
from a healthy human
2. Using a restriction enzyme, cut the
insulin producing gene out of a the DNA
WHAT ARE RESTRICTION ENZYMES?
Bacterial enzymes – used to cut bacteriophage DNA
(viruses that invade bacteria).
Different bacterial strains express different restriction
enzymes
Restriction enzymes recognize a specific short
nucleotide sequence
For example, Eco RI recognizes the sequence:
5’ - G A A T T C - 3’
3’ - C T T A A G - 5’
Pandindrones 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
CTGGCTTAA
GTCAATTAAGCTTAA
Sticky end
AATTC
G
Sticky end - tails
of DNA – easily
bind to other
DNA strands
BLUNT & STICKY ENDS
Sticky ends – Creates an overhang. EcoRI
Blunts- Enzymes that cut at precisely opposite sites
without overhangs. SmaI is an example of an enzyme
that generates blunt ends
CLONING VECTORS
Cloning vector is a carrier that is used to clone a
gene and transfer it from one organism to
another.
Many bacteria contain a cloning vector called a
PLASMID.
PLASMID  is a ring of DNA found in a
bacterium in addition to its main chromosome
3. CUT CLONING VECTOR:
Use bacterial plasmids
Plasmids will be cut with the same restriction
enzyme used to cut the desired gene
4. Ligation - Donor gene (desired gene) is then
spliced or annealed into the plasmid
using DNA ligase as the glue.
Recombinant DNA - DNA with new piece of
genetic information on it
5. Plasmid is then returned to bacterium and
reproduces with donor gene in it.
Transgenic organism – organism with
foreign DNA incorporated in its
genome (genes)
6. Bacterium reproduces and starts producing
human insulin gene which we harvest from them.
Recombinant DNA
Donor Gene
EXPRESSION OF CLONED GENES
Sometimes PROMOTERS must also
be transferred so the genes will be
turned on.
Genes are often turned off until the
proteins they code for are needed.
HOW CAN YOU GET A DESIRED TRAIT WITHOUT
DIRECTLY MANIPULATING THE ORGANISMS
DNA?
1. HYBRIDIZATION; crossing organisms of different traits
to produce a hardier product ex: mule
2. INBREEDING/SELECTIVE BREEDING; maintain the
present genes by breeding only within the population
ex: pedigree animals
3. INDUCING MUTATIONS; radiation, chemicals 
polyploidy (3N or 4N) plants resulted  larger and
hardier
NOW LET US MANIPULATE THE GENES BY
ALTERING THE ORGANISMS DNA
a) DNA Technology: sci. involved in the ability to
manipulate genes/DNA
a) Cure disease
b) treat genetic disorders
c) Improve crops
TOOLS:
1. DNA extraction
2. Restriction enzymes
3. Gel electrophoresis
4. DNA ligase
5. Polymerase chain rxn. (PCR)
METHOD: (5 STEPS)
1.Extract gene  insulin
2.Cut insulin producing gene out using “restriction enzymes”
1.Sticky ends  create overhang
2.Blunts no overhangs
3.Cutting clone vector cut plasmid with same restriction enzyme
4.Ligation: donor gene is spliced into plasmid DNA, DNA ligase
glues it
(this forms recombinant DNA = plasmid DNA + new piece of
DNA)
5.Plasmid returned to bacterium & reproduces using donor gene
in it (this is transgenic organism = organism with foreign DNA
incorporated in it’s genome)
6.*reproduce*
RESTRICTION ENZYMES
BACTERIAL ENZYMES are used to cut DNA
molecule into more manageable pieces
They recognize certain sequences
Creating “single-chain” tails in DNA 
called STICKY ENDS
STICKY ENDS
Readily bind to complimentary chains of
DNA therefore pieces of DNA that have
been cut with the same restriction
enzyme can bind together to form a new
sequence of nucleotides
Recognizes  CTTAAG
*
*
CLONING VECTORS
Cloning vector is a carrier that is used to clone a gene
and transfer it from one organism to another.
Many bacteria contain a cloning vector called a
PLASMID.
PLASMID  is a ring of DNA found in a bacterium in
addition to its main chromosome.
PROCEDURE
To be used as a cloning vector in gene transfer
experiments a plasmid is isolated from a
bacterium.
Using restriction enzymes the plasmid is then
cut and a DONOR GENE (specific gene
isolated from another organism is spliced into
it)
Then the plasmid is returned to the bacterium,
where it is replicated as the bacterium divides,
making copies of the donor gene.
Plasmid now contains a GENE CLONE
*
CLONING VECTORS
!
PLASMID
*
TRANSPLANTING GENES
In some cases, plasmids are used to transfer a gene to
bacteria so that the bacteria will produce a specific
protein
 Ex: INSULIN = protein that controls sugar metabolism
 Bacteria that receives the gene for insulin will produce
insulin as long as the gene is not turned off
STEPS:
1.
ISOLATING A GENE – isolate the DNA from human
cells and plasmids from the bacteria
 Use restriction enzyme
 Splice human DNA into plasmids to create a genomic
library (set of thousands of DNA pieces from a genome
that have been inserted into a cloning vector)
Steps cont…
2.
PRODUCING RECOMBINANT DNA = combination of
DNA from 2 or more sources
 Inserting a donor gene such as human gene for insulin,
into a cloning vector, such as bacterial plasmid results
in a recombinant DNA molecule!
Steps cont…
3. CLONING
DNA – the plasmid containing recomb. DNA
is inserted into a host bacterium (called transgenic
organism
 The trans. Bact. Is placed in a nutrient medium where it
can grow and reproduce.
*
*
EXPRESSION OF CLONED GENES
Sometimes PROMOTERS must
also be transferred so the genes
will be turned on.
Genes are often turned off until
the proteins they code for are
needed.
PRACTICAL USES OF DNA TECHNOLOGY
1. Pharmaceutical products: insulin, HBCF (human blood clotting factor)
2. Genetically engineered vaccines
3. Increased agriculteral yields
4. Improving quality of produce
1.Slow down ripening
2.Enhance color
3.Reduce fuzz
4.Increase flavor
5.Frost resistance
NEGATIVES
Allergies
Label’s don’t include all information
May create “super weeds”
GENE THERAPY
Treatment of genetic disorders
Ex: cystic fibrosis
DNA TECHNOLOGY TECHNIQUES
I.
DNA Fingerprints  pattern of bands made up of
specific fragments from an individual’s DNA
USED FOR:
DETECTION OF A RELATIVE
SIMILARITIES BETWEEN SPECIES
HOW DO YOU MAKE DNA
FINGERPRINTS?
RFLP (restriction fragment length polymorphism)
analysis
1. extract DNA from specimen using restriction
enzymes
2. separate fragments of DNA using
electrophoresis (separates DNA according to size
and charge)
3. placed in wells made on gel and run electric
current through gel
4. blotted onto filter paper/ photgraphic film.
HOW DO YOU MAKE DNA
FINGERPRINTS?
RFLP (restriction fragment length polymorphism)
analysis
 1. extract DNA from specimen using restriction
enzymes
 2. separate fragments of DNA using electrophoresis
(separates DNA according to size and charge)
 3. placed in wells made on gel
 4. electric current run through gel
Continue…
5. negative fragments migrate to
positive charged end of gel but not all at
same rate
6. pores in gel allow smaller fragments
to migrate faster  separating
fragments by size.
7. blotted onto filter paper.
CAN YOU TELL IF THIS COULD BE
THE FATHER?
ACCURACY OF DNA FINGERPRINTS
DNA fingerprints are very accurate
However, genetic tests can only
absolutely disprove, not prove,
relationship!
Courts accept 99.5% accuracy as proof of
alleged paternity
POLYMERASE CHAIN REACTION (PCR)
Used when you only have a TINY piece of
DNA
PCR can be used to quickly make many
copies of selected segments of the
available DNA
Use a PRIMER to initiate replication
DNA doubles every 5 minutes
PCR IS USED FOR:
1. crimes
2. diagnosing genetic disorders
from embryonic cells
3. studying ancient fragments of
DNA (tiny amounts)
HUMAN GENOME PROJECT
2 GOALS:
1. determine nucleotide sequence of
entire human genome (aprox 3 billion
nucleotide pairs or about 100,000
genes
2. map the location of every gene on
each chromosome
1996
1 % of 3 billion nucleotide pairs of
DNA human genomes were
analyzed
This allows for us to identify and
determine the function of 16,000
genes!
GENE THERAPY
Treating a genetic disorder by introducing
a gene into a cell or by correcting a
gene defect in a cell’s genome.
Ex: Cyctic fibrosis cause one defective
gene  malfunction of one protein
GENE THERAPY FOR CYCTIC
FIBROSIS
Nasal spray carrying normal cyctic
fibrosis gene to cells in nose and
lungs
Must repeat treatment periodically
ETHICAL ISSUES
Many people worry about how personal genetic information will be used:
Insurance???
Employment????
Human Genome Project will undoubtedly
involve ethical decisions about how society
should use the information! WHAT DO YOU
THINK??
PRACTICAL USES OF DNA
TECHNOLOGY
1. produce perscription drugs
 Vaccine (harmless version of a virus or a bacterium)
 Pathogen (disease causing agent) treated chemically or
physically so that they can no longer cause disease.
 Pathogen (Ag)  Antibody (Ab)
 DNA tech. may produce vaccines safer than traditional
ones!
INCREASING AGRICULTURAL YIELDS
DNA Tech.  used to develop
new strains of plants
Ex: scientists can make tomato
plants toxic to hornworms and
effectively protect the plant from
these pests.
SEE THE HORNWORM BEGINNING TO
FORM AT THE LEAVES!
THIS HORNWORM EATS AND
DESTROYS THE TOMATO PLANT!
HORNWORMS ATTACK TOMATO PLANTS
CROPS THAT DO NOT NEED
FERTILIZER
Plants require NITROGEN to make
proteins and nucleic acids
Most plants get their N from the soil
TRANSGENIC FOOD CROPS  contain
genes for nitrogen fixation so they
can grow in nitrogen POOR soil.
GENETICALLY ENGINEERED FOODS
Foods may have toxic proteins or
substances  causing ALLERGIES
Ex: changing the gene that codes for an
enzyme to ripening in tomatoes they
are able to make tomatoes ripen
without becoming SOFT!!
GENETICALLY ENGINEERED CROPS
Some are concerned that genetically
engineered crops could spread into
the wild and wipe out native plant
species.
SUPERWEEDS!!!!!!!!