Ch. 13 Genetic EngineeringCh. 13 Genetic Engineering

Ch. 13 OutlineCh. 13 Outline

13-1: Changing the Living World13-1: Changing the Living WorldSelective BreedingSelective Breeding Increasing VariationIncreasing Variation

13-2: Manipulating DNA13-2: Manipulating DNAThe Tools of Molecular BiologyThe Tools of Molecular BiologyUsing the DNA SequenceUsing the DNA Sequence

Ch. 13 OutlineCh. 13 Outline

13-3: Cell Transformation13-3: Cell TransformationTransforming BacteriaTransforming BacteriaTransforming Plant CellsTransforming Plant CellsTransforming Animal CellsTransforming Animal Cells

13-4: Applications of Genetic Engineering13-4: Applications of Genetic EngineeringTransgenic OrganismsTransgenic OrganismsCloningCloning

What is genetic engineering?What is genetic engineering?

In 1973, Mr. Cohen and Mr. Boyer In 1973, Mr. Cohen and Mr. Boyer conducted an experiment on the DNA of conducted an experiment on the DNA of an American frog. They found and an American frog. They found and isolated the gene that codes for ribosomal isolated the gene that codes for ribosomal RNA in the DNA of the frog. They RNA in the DNA of the frog. They removed that gene from the frog and removed that gene from the frog and inserted it into some inserted it into some E. ColiE. Coli Bacteria. Bacteria.

What Happened?What Happened?

During transcription, the bacteria then During transcription, the bacteria then produced the frog RNA!produced the frog RNA!

Genetic Engineering:Genetic Engineering: the process of the process of manipulating (moving) genes for practical manipulating (moving) genes for practical purposes (useful)purposes (useful)

Recombinant DNA: Recombinant DNA: DNA made from 2 or DNA made from 2 or more organisms that are different. more organisms that are different.

The Basic Steps of Genetic The Basic Steps of Genetic EngineeringEngineering

1.1. Cutting the DNA:Cutting the DNA: Restriction Enzymes: bacterial enzymes that Restriction Enzymes: bacterial enzymes that

recognize and bind to specific short recognize and bind to specific short sequences of DNA, and then cute the DNA sequences of DNA, and then cute the DNA between specific nucleotides within the between specific nucleotides within the sequences.sequences.

Vector:Vector: agent used to carry the gene of agent used to carry the gene of interest – usually plasmidsinterest – usually plasmids

PlasmidPlasmid: the circular DNA molecules that : the circular DNA molecules that replicatereplicate

The Basic Steps to Genetic The Basic Steps to Genetic EngineeringEngineering

2.2. Making Recombinant DNAMaking Recombinant DNA DNA fragments of interest (that have already DNA fragments of interest (that have already

been cut) are combined with the vector.been cut) are combined with the vector. DNA ligaseDNA ligase – the enzyme bonds the 2 ends – the enzyme bonds the 2 ends

of the fragments to the vectors.of the fragments to the vectors.

3.3. CloningCloning Gene cloningGene cloning: the process of making many : the process of making many

copies of a genecopies of a gene Bacteria reproduce by binary fissionBacteria reproduce by binary fission

The Basic Steps to Genetic The Basic Steps to Genetic EngineeringEngineering

4.4. ScreeningScreening Cells that have received the gene of interest Cells that have received the gene of interest

are separated out.are separated out. Those cells then continue to produce the Those cells then continue to produce the

protein coded for by the geneprotein coded for by the gene

Cutting DNA & Making Cutting DNA & Making Recombinant DNARecombinant DNA

How Restriction enzymes work:How Restriction enzymes work: The Enzymes recognize specific sequences on The Enzymes recognize specific sequences on

Human and Bacterial PlasmidsHuman and Bacterial Plasmids The Enzymes cut the strands.The Enzymes cut the strands. The cut produces DNA fragments with short strands The cut produces DNA fragments with short strands

on each end that are complementary to each otheron each end that are complementary to each other ““Sticky Ends”Sticky Ends”

Both the human DNA and the Plasmid “Open Up” Both the human DNA and the Plasmid “Open Up” with the same sticky ends remainingwith the same sticky ends remaining

They Bind TogetherThey Bind Together

Diagram

Recognition sequences

DNA sequence

Recognition sequences

DNA sequence

Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end

Confirmation of a Cloned GeneConfirmation of a Cloned Gene

One method used identify a specific One method used identify a specific gene is called a Southern Blotgene is called a Southern Blot

Steps:Steps:1.1. Cut DNA from bacteria with restriction Cut DNA from bacteria with restriction

enzymes.enzymes.

2.2. DNA fragments are separated by a gel DNA fragments are separated by a gel soaked in a chemical solution.soaked in a chemical solution.

Gel electrophoresis – Gel electrophoresis – uses an electric field uses an electric field within a gel to separate molecules by their sizewithin a gel to separate molecules by their size

Confirmation of a Cloned GeneConfirmation of a Cloned Gene

Negatively charged DNA is put into these Negatively charged DNA is put into these wells.wells.They are attracted to the positive pole from They are attracted to the positive pole from

the electric field.the electric field.The Smallest DNA fragments move the The Smallest DNA fragments move the

fastestfastest

Gel Electrophoresis

DNA plus restriction enzyme

Mixture of DNA fragments

Gel

Power source

Longer fragments

Shorter fragments

Confirmation of a Cloned GeneConfirmation of a Cloned Gene

3.3. The DNA separated is then transferred to The DNA separated is then transferred to a filter paper (blotted) and a probe a filter paper (blotted) and a probe solution is added. solution is added.

Probes:Probes: radioactive RNA or single-stranded radioactive RNA or single-stranded DNA pieces that are complementary to the DNA pieces that are complementary to the gene of interestgene of interest

4.4. Only DNA fragments complementary to Only DNA fragments complementary to the probe will form and bind bandsthe probe will form and bind bands

Confirmation of Cloned GenesConfirmation of Cloned Genes

Why do this?Why do this?Bacterial colonies can be used to produce Bacterial colonies can be used to produce

large quantities of the protein (used to study large quantities of the protein (used to study or make drugs)or make drugs)

Genetically engineered Drugs and Genetically engineered Drugs and VaccinesVaccines

Today, many pharmaceutical companies Today, many pharmaceutical companies around the world produce important around the world produce important proteins using genetic engineering.proteins using genetic engineering.

VaccineVaccine: a solution containing all or part : a solution containing all or part of a harmless version of a pathogen; used of a harmless version of a pathogen; used to prevent viral diseases (don’t respond to to prevent viral diseases (don’t respond to drugs)drugs)

Many vaccines are made using genetic Many vaccines are made using genetic engineeringengineering

DNA fingerprintingDNA fingerprinting

DNA fingerprinting: DNA fingerprinting: a pattern of dark bands on a pattern of dark bands on photographic film that is made when an photographic film that is made when an individuals DNA restriction fragments are individuals DNA restriction fragments are separated by gel electrophoresis, probed, and separated by gel electrophoresis, probed, and exposed to X-ray film.exposed to X-ray film.

DNA fingerprints can be used to establish DNA fingerprints can be used to establish paternity, identify genetic disorders, or in paternity, identify genetic disorders, or in forensics (scientific study of cause of injury or forensics (scientific study of cause of injury or death in criminal activity)death in criminal activity)

Improving CropsImproving Crops

Genetic engineers can add favorable Genetic engineers can add favorable characteristics to a plantcharacteristics to a plantResistant to insects (no longer need Resistant to insects (no longer need

pesticides); resistant to weed killer (so crops pesticides); resistant to weed killer (so crops won’t die, but weeds will); improved nutrition – won’t die, but weeds will); improved nutrition – rice + cornrice + corn

Plant Transformation

Recombinant plasmid

Gene to be transferred

Agrobacterium tumefaciens

Cellular DNA

Transformed bacteria introduce plasmids into plant cells

Plant cell colonies

Complete plant is generated from transformed cell

Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome

Animal FarmingAnimal Farming

Growth hormones given to cows to Growth hormones given to cows to produce more milkproduce more milk

Human genes added to farm animals in Human genes added to farm animals in order to have human proteins in their milkorder to have human proteins in their milkThe Human proteins are extracted from milk The Human proteins are extracted from milk

and sold to pharmacy companies.and sold to pharmacy companies.Useful for complex proteins that can’t be made in Useful for complex proteins that can’t be made in

bacteriabacteria

Creating HGH

Human Cell

Gene for human growth hormone

Recombinant DNA

Gene for human growth hormone

Sticky ends

DNA recombination

DNA insertion

Bacterial Cell

Plasmid

Bacterial chromosome

Bacterial cell for containing gene for human growth hormone

Animal FarmingAnimal Farming

Transgenic animalsTransgenic animals: Animals that have foreign : Animals that have foreign DNA in their cellsDNA in their cells

Cloning of animals is another way to make large Cloning of animals is another way to make large quantities of a certain protein.quantities of a certain protein.

How it works: an intact nucleus from an How it works: an intact nucleus from an embryonic cell (whose DNA has recombined embryonic cell (whose DNA has recombined with a human gene) is placed into an egg whose with a human gene) is placed into an egg whose nucleus has been removed. The “new” egg is nucleus has been removed. The “new” egg is then placed into the uterus of an animal.then placed into the uterus of an animal.

CloningA donor cell is taken from a sheep’s udder. Donor

Nucleus

These two cells are fused using an electric shock.

Fused Cell

The fused cell begins dividing normally.

EmbryoThe embryo is placed in the uterus of a foster mother.Foster

Mother

The embryo develops normally into a lamb—Dolly

Cloned Lamb

Egg Cell

An egg cell is taken from an adult female sheep.

The nucleus of the egg cell is removed.