Genetic EngineeringChapter 10

What we are hoping to accomplish•Define genetic engineering.•Explain the principles of genetic

engineering.•Explain and use the tools of genetic

engineering.•Show and describe the practical uses of

genetic engineering.•Discuss the various steps in PCR and

transformation.

Biotechnology-Genetic Engineering•The application of biological and physical

principles to alter the genes of organisms to help solve human problems▫Improving crops and their yield▫Producing insulin in bacteria▫Genetically modified vaccines▫Improving the freshness of fruits and

vegetables

Biotechnology

Historical Aspects

Plant breedingAnimal Breeding

Wine makingDairy products

BreadAntibiotics

Herbal medicine

Business and Industry

BiomedicalAgricultureForensicsIndustrial production

ConservationWildlife

management

Multidisciplinary Research

BiochemistryMolecular genetics

BiophysicsImmunologyCell culture

FermentationBacteriology

BioengineeringGenetic medicine

ConservationEvolutionary

biology

Social and Ethical Implications

Genetic privacyOwnership of

info.Screening

Engineered foodInsuranceTreatment

DNA profilingPatenting of life

What does genetic engineering mean?• It means to isolate a gene from the DNA of one

organism and transfer the gene into the DNA of another.

•We can do this because DNA is DNA in all organisms. It is made out of the same 4 nitrogen bases. The only difference is the sequence.

•From there, DNA codes for RNA and proteins.

Recombinant DNA

•A molecule of DNA made from pieces of DNA from separate organisms.

Restriction Enzyme

•An enzyme that is used to “cut” DNA at certain places along BOTH sides of the DNA.

•Most commonly used Restriction Enzymes▫BamHI- G/GATTC▫HindIII- A/AGCTT▫HpaII- C/CGG▫EcoR1- G/AATTC

Plasmids

•Circular piece of DNA found in bacteria.▫Replicates separate chromosome DNA▫Contains very few genes▫Useful because they have antibiotic

resistance genes on them.▫Typically used as VECTORS to transfer

DNA from one organism to another.

Polymerase Chain Reaction (PCR)•A way to make an unlimited number of

copies of one gene.

•How? DNA Polymerase is used to replicate the same DNA segment over and over.

•Why? ▫Increasing the number (Crime Scenes)▫Sequencing (Prenatal diagnosis)▫Cloning (Woolly Mammoth)

3 Steps to PCR

1. Denaturing- Stands of DNA separate by heating

2. Annealing- Reaction cooled, primers bind

3. Elongation- Reaction heated again, DNA polymerase replicates the rest of the DNA from primer.

•Repeat steps 1-3 about 20-30x

DNA Fingerprinting

•DNA is sliced into fragments called RFLPs.

•DNA is then separated by sequence length by gel electrophoresis.

•The result is for an individual different lengths of DNA that were cut by restriction enzymes that varies among alleles and individuals.

Gel Electrophoresis

How the Gel Works?

•DNA is negative

•When DNA is placed next to a negative charge it will want to move towards the positive charge.

•The heavier strands will not travel as far as the lighter strands.

DNA fingerprinting in a crime scene

Who did it? How Do We Know?

Crime Scene

Suspect 1Suspect 2

Suspect 3

Suspect 4

Use in Crime Scenes

•3 different Fingerprints are run

•1/10,000 chance of matching an individual

•Two fingerprints will only match 1 out of 10,000 X 10,000 times

•Three matching prints would then be 1 in one trillion

The DNA

•Magnified using PCR

•From blood, bone, flesh, hair follicle, semen, saliva, any source of DNA

•Analysis does not compare genes rather the information between genes where a great amount of variation exists ▫RFLP analysis

Restriction Fragment Length Polymorphism (RFLP) Analysis•Identifies locations and numbers of

restriction sites of DNA.

Human Genome Project

•Purpose: Sequence the entire human genome.

•Why: detect, treat, and prevent genetic diseases.

•Completed in 2003•Some of their findings for human DNA

▫Approximately 20,000-25,000 genes (23,000)

▫3 billion base pairs make up the DNA.▫Only 1.5% is actually coding for protein

•Still left to do? Figure out how it all fits together.

Typical Transformation Experiment1. A target gene is isolated and removed by a

restriction enzyme.2. Another restriction enzyme will cut the DNA

of a vector (plasmids) and insert our target gene.

3. Transfer the vector to the organism we want to modify.

4. Transformation- the gene becomes one with its host and replicates producing clone copies.

5. Screen the cells and select modified organism by identifying our gene of interest.

Why do we care?Genetically Engineered Drugs

Genetically Engineered Vaccines

• Humulin - Diabetes• HGH - Dwarfism• Factor VIII, Factor IX -

Hemophilia• t-PA – Heart attack, clot

buster• Lactoferrin – Milk protein

• Herpes II• Hepatitis B• Influenza

Why do we care?Cures for Genetic Diseases Forensic Science

• Cystic fibrosis• Genetic testing• Therapy• Karyotyping

• DNA fingerprints▫ Crime▫ Paternity▫ Predisposed to disease

This may involve using stem cells, by taking somatic cells (like skin cells) and dedifferentiating them down to pluripotent stem cells.

Why do we care?Plants Animals

• Ti plasmid causes plant tumors

• Glyphosate resistant crops (round-up ready)

• Pest resistance• Improve taste and

nutrition• Vaccines grown in plants

• BST• Lactoferrin• Cloning