Download AP genetic technology

Genetic Technology
Genetic Engineering
• Deliberately altering the information content
of DNA molecules
• Genes are isolated, modified, and inserted
into an organism
• Made possible by recombinant technology
– Cut DNA up and recombine pieces
– Amplify modified pieces
Gene Therapy
• The transfer of one or more normal or
modified genes into and individual’s
body cells to correct a genetic defect or
boost resistance to disease
Restriction Enzymes
• Hamilton Smith was studying how Haemophilus
influenzae defend themselves from bacteriophage attack
– Discovered bacteria have an enzyme that chops up
viral DNA
• Restriction enzymes cut DNA at a specific sequence
• Number of cuts made in DNA will depend on number of
times the “target” sequence occurs
• Recombinant DNA can be formed: by splicing 2
different segments of DNA together to create a new
strand
Making Recombinant DNA
•Recombinant DNA can be formed: by splicing 2 different
segments of DNA together to create a new strand
5’
G
A A T T C
3’
C T T A A
G
one DNA fragment
another DNA fragment
5’
G
A A T T C
3’
3’
C
T T A A
5’
G
In-text
figure
Page 254
Making Recombinant DNA
nick
5’
G A A T T C
3’
3’
C
5’
T T A A
G
nick
DNA ligase action
G A A T T C
C T T A A G
In-text
figure
Page 254
Using Plasmids
• Plasmid is small circle of bacterial DNA
• Foreign DNA can be inserted into
plasmid
– Forms recombinant plasmids
– Plasmid is a cloning vector
– Can deliver DNA into another cell
Using Plasmids
DNA
fragments
+
enzymes
Figure 16.4
Page 255
recombinant
plasmids
host cells containing
recombinant plasmids
mRNA transcript
Making cDNA
•cDNA is a DNA
molecule copied from a
mature mRNA
transcript by reverse
transcription
Figure 16.5
Page 255
mRNA–cDNA hybrid
single-stranded cDNA
double-stranded cDNA
Amplifying DNA
• Ways to take small pieces of DNA and
make them larger
– Fragments can be inserted into
fast-growing microorganisms
– Polymerase chain reaction (PCR)
Polymerase Chain Reaction
• Sequence to be copied is heated
• Primers are added and bind to ends of
single strands
• DNA polymerase uses free nucleotides
to create complementary strands
• Doubles number of copies of DNA
Polymerase
Chain
Reaction
Double-stranded
DNA to copy
DNA heated to
90°– 94°C
Primers added to
base-pair with
ends
Mixture cooled;
base-pairing of
primers and ends
of DNA strands
Stepped Art
Figure 16.6
Page 256
DNA polymerases
assemble new
DNA strands
Polymerase
Chain
Reaction
Mixture heated again;
makes all DNA
fragments unwind
Mixture cooled; basepairing between
primers and ends of
single DNA strands
Stepped Art
Figure 16.6
Page 256
DNA polymerase
action again
doubles number of
identical DNA
fragments
DNA Fingerprints
• Individuals have a unique array of DNA
fragments
• Inherited from parents in Mendelian
fashion
• Even full siblings can be distinguished
from one another by this technique
Tandem Repeats
• Short regions of DNA that differ
substantially among people
• Many sites in genome where tandem
repeats occur
• Each person carries a unique
combination of repeat numbers
RFLPs
• Restriction fragment length polymorphisms
• DNA from areas with tandem repeats is cut
with restriction enzymes
• Because of the variation in the amount of
repeated DNA, the restriction fragments
vary in size
• Variation is detected by gel electrophoresis
Gel Electrophoresis
• DNA is placed at one end of a gel
• A current is applied to the gel
• DNA molecules are negatively charged
and move toward positive end of gel
• Smaller molecules move faster than larger
ones
Analyzing DNA Fingerprints
• DNA is stained or made visible by use
of a radioactive probe
• Pattern of bands is used to:
– Identify or rule out criminal suspects
– Identify bodies
– Determine paternity
Genome Sequencing
• 1995 - Sequence of bacterium
Haemophilus influenzae determined
• Automated DNA sequencing now main
method
• Draft sequence of entire human
genome determined in this way
• Human Genome Project
The Human Genome Initiative
Goal - Map the entire human genome
• Initially thought by many to be a waste
of resources
• Process accelerated when Craig
Ventner used bits of cDNAs as hooks to
find genes
• Sequencing was completed ahead of
schedule in early 2001
Genomics
• Structural genomics: actual mapping
and sequencing of genomes of
individuals
• Comparative genomics: concerned with
possible evolutionary relationships of
groups of organisms
Gene Libraries
• Bacteria that contain different
cloned DNA fragments
– Genomic library
– cDNA library
Using a Probe to Find a Gene
Colonies on plate
• You want to find which bacteria in
Cells adhere
to filter
a library contain a specific gene
• Need a probe for that gene
Cells are lysed;
DNA sticks
to filter
– A radioisotope-labeled piece of
DNA
– Will base-pair with gene of
interest
Probe is
added
Location where probe binds forms
dark spot on
film,
indicates
colony with
gene
Cloning
•Making a genetically identical copy of DNA
or of an organism
•Three different types
–Recombinant DNA or DNA cloning (we already
talked about)
–Reproductive Cloning – making of an entire
organism
–Therapeutic Cloning – also called “embryo
cloning”; stem cells
Cloning Dolly
1997 - A sheep cloned from an adult cell
– Nucleus from mammary gland cell was
inserted into enucleated egg
– Embryo implanted into surrogate
mother
– Sheep is genetic replica of animal from
which mammary cell was taken
Engineered Proteins
• Bacteria can be used to grow medically
valuable proteins
– Insulin, interferon, blood-clotting
factors
– Vaccines
Cleaning Up the Environment
• Microorganisms normally break down
organic wastes and cycle materials
• Some can be engineered to break down
pollutants or to take up larger amounts
of harmful materials
Engineered Plants
• Cotton plants that display resistance to
herbicide
• Aspen plants that produce less lignin
and more cellulose
• Tobacco plants that produce human
proteins
• Mustard plant cells that produce
biodegradable plastic
First Engineered Mammals
• Experimenters used mice with hormone
deficiency that leads to dwarfism
• Fertilized mouse eggs were injected
with gene for rat growth hormone
• Gene was integrated into mouse DNA
• Engineered mice were 1-1/2 times
larger than unmodified littermates
Can Genetically Engineered
Bacteria “Escape”?
• Genetically engineered bacteria are
designed so that they cannot survive
outside lab
• Genes are included that will be turned
on in outside environment, triggering
death
Ethical Issues
• Who decides what should be “corrected”
through genetic engineering?
• Should animals be modified to provide
organs for human transplants?
• Should humans be cloned?