IB Genetics Topic 4:

Genetic Engineering

Topics in Genetic Engineering

• Polymerase Chain Reaction (PCR)• Gel electrophoresis• DNA profiling

• The genome project• Gene transfer

• Genetic modification in plants and animals• Cloning

We will be brief!

• The Blog has videos and games for you to cement your understanding of each topic!

4.4.1: The Polymerase Chain Reaction

• Kary Mullis, 1984, Nobel Prize 1993

• ‘Reviewers and Biographers often latch on to his enjoyment of drugs, womanising and surfing to paint him as some sort of cool rock star rebel…but…’

What does PCR do?

• It quickly amplifies a tiny sample of DNA into millions of identical copies

• This produces enough DNA to allow further analysis and study.

The DNA sequences amplified by PCR are used for:

• Forensic identification - crime (CSI….)

• Paternity suits• The Genome project: determination

of the entire human genetic code• Diagnosing diseases and genetic

disorders – genotype analysis• Detection of bacteria and viruses in

the environment• Evolutionary research

How does PCR work?

First, you need to extract DNA from cells.Next, PCR requires:• Custom-made DNA primers• (TAQ) DNA polymerase• Nucleotides for complementary

base pairing• HEATING and COOLING:

Thermocycler machinePCR can produce 100 billion copies of the DNA sample in a few hours

Let’s do it!• PCR the movie• PCR the cartoon• PCR the game

4.4.2: Gel electrophoresis1. Commonly the DNA sample is first amplified

by PCR2. The DNA sample is chopped into fragments

using restriction enzymes3. Gel electrophoresis separates fragments of

DNA according to their size4. DNA samples are placed in a gel,

fluorescent marker added (tag attached to a triplet), and an electric charge is applied to push fragments along

5. The shortest fragments travel furthest through the gel, while the longest (heaviest) remain closer to their origin

6. This lets us ‘match’ the sample against another DNA sample or standard

Let’s do it!

• Virtual Lab 1

• Virtual lab

4.4.3: DNA profiling

HOW?• DNA sample (blood, semen,

cheek swab)• Sample is amplified by PCR,

then matched against known samples using gel electrophoresis

• We look for standard tandem repeating DNA: highly repetitive sequences from non-coding region of DNA

• STR’s are unique to individuals

APPLICATIONS

Numerous techniques for DNA profiling:• Criminal forensics• Paternity suits• Ecology – identifying

relationships in migrating birds, whales etc

• Evolutionary genetics

Famous examples of DNA profiling

• The innocence project• The famous blue dress• Identifying the Mona Lis

a• Identifying the Romano

vs

DNA profiling: paternity and forensics

• The fluorescent banding pattern shows up for

each DNA fragment and can be compared with

test/ known sample• Usually >4 ‘STR’ sites

are used to establish paternity or identity

4.4.6: The Human Genome project

• We’re all the same!

• We’re all different!• Welcome to the human genome

Started in 1990, first draft published in 2003, ongoing refinement…Amazing international effort• The BBC tell us about the genome 10 years on• Hank tells us some surprises about the human genome

project• ..so what has the human genome project achieved, so fa

r?

Key outcomes of the Human Genome project

• We identified the number (30,000) and loci of all the genes of our genome

1. Human health: Medical diagnostics, treatments, pharmacogenomics, gene therapy• We identified many new proteins and their functions2. Transgenics: move production of beneficial proteins from one species to another, design novel proteins • We compared human DNA with that of other species3. Evolutionary genetics and evolutionary history4. Bio-informatics: Genetic databases and the bio-informatics industry

The HGP and human health

• 2 out of 3 of us will die of a genetically related disease

• Many (some say all) diseases have a genetic cause

• Genetic report cards can identify ‘potential risk’ of future illnesses

• The future is here...

Do you want to know your genome?

• Do you want to know your genome?

• Are you sure?

The human genome and new medicine (1): Gene therapy

• Find defective genes and ‘fix them’

• Insert a healthy normal gene or gene product (lipid, protein)to achieve normal function

• Most effective for single gene disorders: cystic fibrosis, Sickle Cell disease, • gene therapy in depth

• A lucrative business...

The human genome provides avenues for new medicines

• Identify beneficial molecules which are produced in healthy people

• Identify the gene which produces the desirable molecule

• Copy that gene and use it as a recipe to synthesise the molecule in a laboratory (or in another organism!)

• Distribute the beneficial molecule as a new treatment

The HGP and medicine (3): Pharmacogenomics

Uses information about your genetic make-up to determine the drug, and drug doses, that will work best for you

Currently used for treatment of:• HIV• Breast cancer• Colon cancer• Mental illnessmore info here...

3. Using the HGP to understand human evolution

By sequencing and databasing genes, we can see similarities and differences between species• The closer the genome match, the

closer their evolutionary history• Human Chromosome 2 came

from fusion of two great ape chromosomes

• Karl Miller on human evolution• The time-tree of evolution

Ethics and the human genome

How companies are patenting your genetic code

4.4.8: GENETIC ENGINEERING, (MODIFICATION, TRANSGENICS) BIOTECHNOLOGY

The basic premise for genetic engineering…

The genetic code is universal• All living things share the same code• Each codon codes for the same animo aacid,

regardless of the species• Genes can be transferred from species to

species, to produce the same productWe can cut, copy and paste DNA between species

Gene transfer 101: the basics of cutting, copying, pasting and cloning genes

• An introduction• Step-by-step

How does genetic engineering work?

1. Cut out desired DNA using restriction endonuclease

2. Prepare plasmid using the same restriction enzyme

3. Insert DNA into plasmid using DNA ligase

4. Insert plasmid (vector) into host cell

Applied gene transfer: Gene Therapy

Gene therapy for treatment of Severe Combined Immunodeficiency Disease(2)

Genetic Engineering: Making Human insulin

• E. Coli make human insulin

Genetic Engineering: Making Factor IX in sheep…

1997: Factor IX isolated and purified from sheep’s milk to treat one hereditary form of haemophilia (Haemophilia B,

Christmas Disease)

Polly and Molly the Factor IX sheep

Transgenic (GM) plantsConferred Trait Organism Genetic change

Herbicide resistance Soybean ‘Roundup’: Glysophate resistance

Flavr Savr Tomato Switch off gene for ripening – delays natural softening, improves flavour

Insect Resistance Bt Corn Resistance to European corn borer introduced by addition of toxin from Bacillus Thuringiensis

Salt-resistance Tomato Extended range for tomato production

Beta carotene ‘Golden rice’ Protection against beta-carotene deficiency

Transgenic plants: The controversies…

The controversies….

Transgenic (GM) animalsConferred Trait Sheep Genetic changeFluorescence (gfp gene)

Fish (pigs, cats) Fluorescence used as biosensor for other transferred genes

Factor IX production

Sheep Production of Factor IX in milk for haemophilia

Milk quality Cows Milk equivalent to human milk

Glowing animals?....what’s the big deal?

Worth a Nobel prize….

Further Applications of Transgenic Technology

• Vaccines (transgenic yeast produces vaccine for Hepatitis B)

• Transgenic mice widely used for human disease research

• Transgenic male mosquitoes carrying ‘lethal gene’ used to reduce the incidence of Dengue fever

Genetic modification: the PROs…• GM crops should improve yields,

quality and food security• Pest-resistant crops will reduce the

need for pesticides• GM can produce crops which provide

dietary supplements such as retinol• GMO’s used to produce rare proteins

for medicine or vaccines will be cheaper and less polluting than conventional methods

• GM allows farmers greater control for selective breeding of crops and animals

Genetic modification: the CONs…• No one knows the long-term effects of GMO’s in the biosphere

PRECAUTIONARY PRINCIPLE• Genes from GM crops are easily integrated into the wild type crop• Genes may be able to cross species• Crops which produce toxins to kill insects could prove toxic to

humans• Large portions of the human food supply may fall under the control

of a small number of companies• High-tech agricultural ones are not necessarily better than simpler

solutions• A proliferation of GMO’s could drastically reduce natural

biodiversity

Where do you stand?

4.4.11: CLONING

What is a clone?

• A group of genetically identical organisms• A group of cells artificially derived from a

single parent

Natural Clones

• Asexual reproduction: bacteria, yeasts, protozoa

• Vegetative propagation in plants

• Monozygotic (identical) twins

4.4.12: ‘Outline a technique for cloning using differentiated animal

cells’

4.4.13: THERAPEUTIC CLONING: Somatic cell

nuclear transfer

Therapeutic cloning

Ethical issues surrounding therapeutic cloning

• Therapeutic cloning allows production of embryonic stem cells from differentiated cells

• These embryonic stem cells have great potential for a patient to replace their own damaged cells - skin, heart, kidney, brain, etc etc.

• A promising technique of stem cell production

• These embryonic stem cells have the potential to become a human being (so called reproductive cloning)

• Reproductive cloning of humans is illegal in most countries

Therapeutic cloning is aimed NOT at making people, but at CURING

people

Therapeutic cloning: The controversies

1. New potential therapies for disease

2. Production of immuno-compatible tissue for self-transplantation

3. Advances in medical research

4. Cost-benefit firmly in favour of therapeutic cloning

1. Manipulation and destruction of human embryos is wrong

2. Reproductive cloning becomes more likely

3. We will create a global market for women’s eggs