Download Chapter 11: Gene Technology

Chapter 11: Gene
Technology
Biology II
History of Genetic Engineering

In 1973, Stanley Cohen and Herbert Boyer
isolated genes that code for rRNA from DNA of
African clawed frog
 Inserted this DNA into the DNA of E.coli
 E.coli produced frog rRNA during
transcription, producing the first genetically
altered organism.
Basic Steps of Genetic
Engineering


Genetic engineering – the process of
manipulating genes for practical purposes
 May involve building recombinant DNA
– DNA made from 2 or more different
organisms
Can follow the steps of genetic
engineering by examining how human
gene for insulin is transferred into bacteria
Step 1: Cutting DNA

DNA of interest is cut by restriction enzymes
 Bacterial enzymes that recognize and bind to
specific short sequences of DNA and then cut
the DNA between specific nucleotides within
the sequences
 Also cut is the vector – agent used to carry
the gene of interest into another cell
 Commonly used vectors include viruses,
yeast, and plasmids – circular DNA
molecules that can replicate independently
of the main chromosomes of bacteria
Step 2: Making Recombinant
DNA

DNA fragments from organism
containing gene of interest are
combined with DNA fragments from
the vector
 Enzyme called DNA ligase added to
help bond ends of DNA fragments
together
Step 3: Cloning

Many copies of the
gene of interest are
made each time the
host cell reproduces
in process called
gene cloning
 Bacteria reproduce
by binary fission,
producing identical
offspring
Step 4: Screening

Cells that have received gene of
interest are distinguished from those
that did not take up the vector with
the gene of interest
Restriction Enzymes:
Cutting DNA

Restriction enzymes recognize a specific
sequence of DNA
 This sequence and the sequence on the
complementary DNA strand are palindromes
– they read the same forwards and
backwards
 Cuts of most restriction enzymes produce
pieces of DNA with short single strands on
each end, called sticky ends, that are
complementary to each other
Making Recombinant DNA

Vectors used in genetic engineering
contain only one nucleotide sequence
that restriction enzyme recognizes
 Vectors “open up” with same sticky
ends as those of cut human DNA
 2 DNA molecules bond together
through complementary base
pairing of sticky ends
Screening of Engineered Cells


Cells that have taken up plasmid must be
identified
 Accomplished by growing bacteria on plates
that contain the antibiotic tetracycline
 Cells that have taken up the vectors contain
the gene for tetracycline resistance and
therefore survive on this medium
Surviving cells make copy of vector, eventually
forming a colony of genetically identical cells, or
clones
Confirmation of Cloned Genes


Surviving bacterial colonies must be tested for
presence of gene of interest
The Southern Blot
 DNA from each bacterial clone colony is
isolated and cut into fragments by restriction
enzymes
 DNA fragments separated by gel
electrophoresis
 Uses
electric field within gel to separate
molecules by size
Gel Electrophoresis

Gel is rectangular slab of gelatin with line of
rectangular wells near top edge
 DNA sample placed into wells
 DNA is negatively charged so it migrates
towards positive pole when electric field is
applied
 Speed at which DNA
fragments migrate down
well determined by size
(weight), with smallest
moving the fastest
The Southern Blot (continued)


DNA bands transferred (blotted) directly
onto filter paper
 Filter paper moistened with probe
solution – radioactive or fluorescentlabeled RNA or single-stranded DNA
pieces that are complementary to the
gene of interest
Only DNA fragments complementary to
probe will bind and form visible bands
What’s Next?

Bacterial colonies containing gene of interest
can be used in a variety of ways:
 Isolate gene of interest to get pure DNA
 Study evolution of gene by comparison
across different organisms
 Transfer isolated gene of interest to other
organisms
 Produce large quantities of protein coded for
by gene for further study or to make drugs
Section 11-2
Human Applications of Genetic
Engineering
The Human Genome Project

A research effort to sequence and locate the
entire collection of genes in human cells
 Many surprising findings:
 Only 1-1.5% of DNA in human genome
codes for protein
 Human cells contain only 20,000-25,000
genes even though over 120,000 different
forms of mRNA molecules had been
counted
Genetically Engineered Drugs

Drugs can be manufactured by genetic
engineering illnesses that result when body fails
to make critical proteins
 Factor VIII – protein that promotes blood
clotting
 Deficiency in factor VIII causes a type of
hemophilia
 Prior to GM medicines, hemophiliacs
received blood factors isolated from donor
blood, creating many potential risks for
patients
Vaccines

Vaccine – solution containing all or part of
a harmless version of a pathogen
 Once injected, immune system
recognizes pathogen’s surface proteins
and responds by making antibodies
 If future exposure occurs, antibodies are
present to combat pathogen
Genetically Engineered Vaccines


Vaccines traditionally prepared using killed or
weakened forms of pathogen
 Potential to transmit disease if there is failure
in process
GM vaccines are both effective and safe
 Genes that code for surface protein of
pathogen are inserted into DNA of harmless
virus
 Surface of modified virus display the surface
proteins of the pathogen in addition to the
virus’s own surface protein
RFLPs

No 2 individuals (except twins) have the same
DNA sequence
 When cut with restriction enzyme, length of
resulting fragments will be different for each
individual, called restriction fragment length
polymorphism (RFLPs)
 RFLPs used to identify individuals and
determine relatedness among individuals
DNA Fingerprinting

DNA fingerprint – pattern of dark bands on
photographic film made when an individual’s
DNA restriction fragments are separated by gel
electrophoresis, probed, and then exposed on
X-ray film
 Each individual (except twins) will have
unique pattern of banding, or DNA fingerprint,
since they have different RFLPs
Purposes of DNA Fingerprinting
To establish relatedness – paternity
cases
 Forensics – scientific investigation of
of causes of injury and death
 Identifying genes that cause genetic
disorders

PCR

Polymerase Chain Reaction (PCR) – technique
that makes many copies of selected segments
of DNA
 Useful when only a very small amount of
DNA is available
 Can produce a billionfold increase in DNA
material within a few hours
 Important for diagnosing genetic disorders
and solving crimes, as well as for studying
ancient fragments of DNA found in fossils
PCR Technique




Double-stranded DNA to be copied is heated,
separating the strands
Short pieces of artificially made DNA called
primers are added, binding to places of DNA
where copying can begin
DNA polymerase and free nucleotides added,
extending DNA by attaching complementary
free nucleotides to primer
Process repeated, with the sample of DNA
doubling every 5 minutes
Section 11-3
Genetic Engineering in
Agriculture
Genetically Engineered Crops

Genetic engineers can change plants
 Make plant more tolerable to drought
conditions
 Create plants that can adapt to different
soils, climates, and environmental
stresses
 Create crops resistant to weedkiller
glyphosate
Genetically Engineered Crops
(con’d)
 Develop
crops resistant to insects
 Insert gene isolated from soil
bacteria that makes protein that
injures gut of chewing insects
 Make more nutritious crops
 Add iron and beta carotene to rice
Risks of GM Plants

There are some potential problems with GM
crops:
 Weeds that have developed resistance to
weedkillers
 Allergies to products of introduced genes
 Passing on introduced genes to wild relative
 Ex. Wild corn
 Pest resistant to GM toxins
Genetic Engineering in Farm
Animals

Farmers use gene technology to improve or
modify their farm animals:
 Add Growth Hormone (GH) to supplement
diet
 Produced by bacteria into which the gene
was introduced
 Alter gene responsible for GH production in
animals
Medically Useful Proteins


Genetic engineers have been able to add
human genes to the genes of farm animals
 Transgenic animal – animal with foreign
DNA in their cells
 Farm animals then produce human
proteins in milk
Scientists can also clone animals in order to
create herds that can make medically useful
proteins
Cloning From Adult Animals

Ian Wilmut proved that animals could be cloned
from differentiated cells of an adult animal
 Lamb cloned from nucleus of mammary cell
taken from adult sheep
 Electric shock used to fuse mammary cells
from one sheep with eggs cells lacking nuclei
from another sheep
 Fused cells divide to form embryos to be
implanted into surrogate mothers
Problems With Cloning


Only a few cloned animals have survived for
long
 Some fatally oversized
 Others have developmental problems
Genomic imprinting – chemical changes made
to DNA prevent a gene’s expression without
altering its sequence
 Technical problems with this process, since
normal process takes months for sperm and
years for eggs