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’
G
3’
C T T A A
A A T T C
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
recombinant
plasmids
Figure 16.4
Page 255
host cells containing
recombinant plasmids
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
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

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
 cDNA
library
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 tumorcausing genes with
beneficial genes
Plasmid transfers
these genes to
cultured plant cells
plant cell
foreign gene
in plasmid
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
 Sheep
implanted into surrogate mother
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?