Lecture 11Biotechnology

A Scientific Revolution

Genetic engineering is the process of moving genes from one organism to another Having a major impact on agriculture & medicine

Restriction Enzymes

Restriction enzymes bind to specific short sequences (usually 4- to 6- bases long) on the DNA The nucleotide sequence on both

DNA strands is identical when read in opposite directions

Most restriction enzymes cut the DNA in a staggered fashion This generates “sticky” ends

These ends can pair with any other DNA fragment generated by the same enzyme

The pairing is aided by DNA ligase

GAATTCCTTAAG

Play Restriction Enzymes

4 Stages of a Genetic Engineering Experiment

1. Cleaving DNA

2. Producing recombinant DNA

3. Cloning

4. Screening

All gene transfer experiments share four distinct stages

Play Steps in cloning a gene

Stage 1

Cleaving the DNA The large number of fragments produced are separated by

electrophoresis

Fragments appear as

bands under fluorescent light

Stages 2 & 3

Producing Recombinant DNA Fragments of source DNA are inserted into vectors

Vectors are plasmids or viruses that carry foreign DNA into the host cell

Vector DNA is cut with the same enzyme as the source DNA, thus allowing the joining of the two

Cloning Host cells are usually bacteria

As each bacterial cell reproduces, it forms a clone of cells containing the fragment-bearing vector

Together all clones constitute a clone library

Stage 4

Screening

A preliminary screen of the clone library eliminates1. Clones without vectors2. Clones with vectors

that do not contain DNA

The vector employed usually has genes for Antibiotic resistance

This eliminates the first type of clones because they are sensitive to antibiotics

-galactosidase This eliminates the

second type of clones based on X-gal metabolism and color changes

Stage 4 (cont.)

Screening To find the gene of interest, the clone library is screened by a process termed

hybridization The cloned genes form base pairs with complementary sequences on another

nucleic acid, termed the probe

The bacterial colonies are first grown on agar They are then transferred to a filter The filter is treated with a radioactive probe The filter is then subjected to autoradiography

Working with DNA

Key techniques used by today’s genetic engineers include

PCR amplification Used to increase the amounts of DNA

cDNA formation Used to build genes from their mRNA

DNA fingerprinting Used to identify particular individuals

The polymerase chain reaction (PCR) requires primers Short single-stranded

sequences complementary to regions on either side of the DNA of interest

PCR consists of three basic steps

1. Denaturation2. Primer annealing3. Primer extension

PCR AmplificationTarget sequence

Primers

Denaturation1

Heat

2 Annealing of primers

Cool

2 copies Free nucleotides

3 Primer extension

DNA polymerase

Cycle1

Heat

Cool4 copiesCycle

2

Cool

Heat

8 copiesCycle

3

Play Polymerase Chain Reaction

The primary mRNA transcript contains exons and introns

The processed mRNA contains only exons

It is used as a template to create a single strand of DNA termed complementary DNA (cDNA)

cDNA is then converted to a double-stranded molecule

cDNA Formation

This is a process that is used to determine if two DNA samples are from the same source

The DNA from the two sources is fragmented using restriction enzymes The fragments are

separated using gel electrophoresis

They are transferred to a filter

The filters are screened with radioactive probes

Then subjected to autoradiography

DNA Fingerprinting

Play DNA Fingerprinting

Genetic Engineering and Medicine

Genetic engineering has been used in many medical applications

1. Production of proteins to treat illnesses2. Creation of vaccines to combat infections3. Replacement of defective genes

In diabetes, the body is unable to control levels of sugar in the blood because of lack of insulin

Diabetes can be cured if the body is supplied with insulin

The gene encoding insulin has been introduced into bacteria

Other genetically engineered drugs include

Anticoagulants Used to treat heart attack

patients Factor VIII

Used to treat hemophilia Human growth hormone (HGH)

Used to treat dwarfism

Making “Magic Bullets”

Genetic engineering has also been used to create subunit vaccines against viruses

Piggyback Vaccines

A gene encoding a viral protein is put into the DNA of a harmless virus and injected into the body

The viral protein will elicit antibody production in the animal

A novel kind of vaccine was introduced in 1995

The DNA vaccine uses plasmid vectors

It elicits a cellular immune response, rather than antibody production

Genetic Engineering of Farm Animals

In 1994, the recombinant hormone bovine somatotropin (BST) became commercially available Dairy farmers used BST as a supplement to

enhance milk production in cows

Consumers are concerned about the presence of the hormone in milk served to children This fear has not been supported by research data

Genetic Engineering of Crop Plants

Pest resistance Leads to a reduction in the

use of pesticides Bt, a protein produced by

soil bacteria, is harmful to pests but not to humans

The Bt gene has been introduced into tomato plants, among others

Herbicide resistance Crop plants have been

created that are resistant to glyphosate

Herbicide resistance offers two main advantages Leads to a reduction in the use of pesticides Lowers the cost of producing crops Reduces plowing and conserves the top soil

Genetic Engineering of Crop Plants

More Nutritious Crops Worldwide, two major deficiencies are iron and vitamin A Deficiencies are especially severe in developing countries where the

major staple food is rice Ingo Potrykus, a Swiss bioengineer, developed transgenic “golden”

rice to solve this problem

The promise of genetic engineering is very much in evidence

However, it has generated considerable controversy and protest

Are genetic engineers “playing God” by tampering with the genetic material?

Two sets of risks need to be considered

1. Are GM foods safe to eat? The herbicide glyphosate blocks the synthesis of aromatic amino acids

Humans don’t make any aromatic amino acids, so glyphosate doesn’t hurt us

However, gene modifications that render plants resistant to glyphosate may introduce novel proteins Moreover, introduced proteins may cause allergies in humans

2. Are GM foods safe for the environment? Three legitimate concerns are raised

1. Will other organisms be harmed unintentionally?2. Will pests become resistant to pesticides?3. What if introduced genes will pass from GM crops to their wild or

weedy relatives?

Potential Risks of Genetically Modified (GM) Crops

Potential Risks of Genetically Modified (GM) Crops

Should GM foods be labeled?

Every serious scientific investigation has concluded that GM foods are safe

So there is no health need for a GM label

However, people have a right to know what is in their food

So there may be a need for label after all

Cloning Higher Organisms

The successful embryos (about 30 in 277 tries) were transplanted into surrogate mother sheep

On July 5, 1996, “Dolly” was born Only 1 of 277 tries succeeded However, Wilmut proved that reproductive cloning is possible

Since Dolly, scientists have successfully cloned sheep, mice, cattle, goats and pigs However,

problems and complications arise, leading to premature death

Dolly died in 2002, having lived only half a normal sheep life span

Embryonic Stem Cells

The blastocyst, an early embryo, consists of A protective outer layer that

will form the placenta Inner cell mass that will

form the embryo The inner cell mass consists of

embryonic stem cells These are pluripotent

Capable of forming the entire organism

As development proceeds, cells lose their pluripotency They become committed to

one type of tissue They are then called adult

stem cells

The research in human embryonic stem cells is associated with two serious problems Finding a source: harvesting them from

discarded embryos raises ethical issues Immunological rejection: Implanted stem

cells will likely be rejected by the immune system of the individual

Stem Cells

Embryonic stem cells could be used to restore tissues lost or damaged due to accident or disease Experiments have

already been tried successfully in mice

Damaged spinal neurons have been partially repaired

The course of development is broadly similar in all mammals Therefore, the

experiments in mice are very promising

Grappling with the Ethics of Stem Cell Research

Stem cells offer enormous promise for treating a wide range of diseases However, the research involves ethical issues

1. Destruction of human embryos When does human life begin?

2. Possibility of future abuse or misuse Is human reproductive cloning next?

3. Alternative sources of stem cells Are adult stem cells equally effective?

Gene Therapy

Gene therapy involves the introduction of “healthy” genes into cells that lack them

It was first used successfully in 1990 Two girls were cured of a

rare blood disorder caused by a defective adenosine deaminase gene

The girls stayed healthy

In 1999, AAV successfully cured anemia in rhesus monkeys

AAV was also used to cure dogs of a hereditary disorder leading to retinal degeneration & blindness