Recombinant DNA
and Genetic
Engineering
Chapter 16
Familial Hypercholesterolemia
Gene encodes protein that serves as cell’s LDL
receptor
Two normal alleles for the gene keep blood level
of LDLs low
Two mutated alleles lead to abnormally high
cholesterol levels & heart disease
Example of Gene Therapy
Woman with familial hypercholesterolemia
Part of her liver was removed
Virus used to insert normal gene for LDL receptor
into cultured liver cells
Modified liver cells placed back in patient
Results of Gene Therapy
Modified cells alive in woman’s liver
Blood levels of LDLs down 20 percent
No evidence of atherosclerosis
Cholesterol levels remain high
Remains to be seen whether procedure will
prolong her life
Genetic Changes
Humans have been changing the genetics of
other species for thousands of years
Artificial selection of plants and animals
Natural processes also at work
Mutation, crossing over
Genetic Engineering
Genes are isolated, modified, and inserted into
an organism
Made possible by recombinant technology
Cut DNA up and recombine pieces
Amplify modified pieces
Discovery of Restriction Enzymes
Hamilton Smith was studying how
Haemophilus
influenzae
defend themselves from bacteriophage
attack
Discovered bacteria have an enzyme that chops
up viral DNA
Specificity of Cuts
Restriction enzymes cut DNA at a specific
sequence
Number of cuts made in DNA will depend
on number of times the “target” sequence
occurs
Making Recombinant DNA
5’
3’
G
C T T A A
A A T T C
G
G
A A T T C
C T T A A
G
3’
5’
one DNA fragment
another DNA fragment
3’
5’
In
-
text
figure
Page 254
Making Recombinant DNA
nick
5’
3’
3’
5’
G
A A T T C
C T T A A
G
nick
G
A A T T C
C T T A A
G
DNA ligase action
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
recombinant
plasmids
host cells containing
recombinant plasmids
Figure 16.4
Page 255
Amplifying DNA
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
DNA polymerases
assemble new
DNA strands
Figure 16.6
Page 256
Stepped Art
Polymerase
Chain Reaction
Figure 16.6
Page 256
Stepped Art
Mixture heated again;
makes all DNA
fragments unwind
Mixture cooled; base
-
pairing between
primers and ends of
single DNA strands
DNA polymerase
action again
doubles number of
identical DNA
fragments
DNA Fingerprints
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
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
Gene Libraries
Bacteria that contain different
cloned DNA fragments
Genomic library
cDNA library
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
The Ti plasmid
Researchers
replace tumor
-
causing genes with
beneficial genes
Plasmid transfers
these genes to
cultured plant cells
foreign gene
in plasmid
plant cell
Figure 16.11
Page 261
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
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
Designer Cattle
Genetically identical cattle embryos can be
grown in culture
Embryos can be genetically modified
create resistance to mad cow disease
engineer cattle to produce human serum
albumin for medical use
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
Using Human Genes
Even with gene in hand it is difficult to
manipulate it to advantage
Viruses usually used to insert genes into
cultured human cells but procedure has
problems
Very difficult to get modified genes to work
where they should
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?
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