Ch. 13 Genetic Engineering

Ch. 13 Outline 13-1: Changing the Living World 13-2: Manipulating DNA Selective Breeding Increasing Variation 13-2: Manipulating DNA The Tools of Molecular Biology Using the DNA Sequence

Ch. 13 Outline 13-3: Cell Transformation Transforming Bacteria Transforming Plant Cells Transforming Animal Cells 13-4: Applications of Genetic Engineering Transgenic Organisms Cloning

Selective Breeding Humans use selective breeding, which takes advantage of naturally occurring genetic variation in plants, animals, and other organisms, to pass desired traits on to the next generation of organisms

Selective Breeding Nearly all domestic animals -- including horses, cats, and farm animals – and most crop plants have been produced by selective breeding

Hybridization Louis Burbank was the greatest selective breeder of all time. He developed the disease-resistant potato and more than 800 varieties of plants.

Louis Burbank used the technique of hybridization and bred dissimilar individuals to combine the best traits of both parents. The hybrids produced by these crosses were hardier than their parents

Inbreeding To maintain the desired characteristics of a line of organisms, breeders often use the technique of inbreeding. Inbreeding is the continued breeding of individuals with similar characteristics

Increasing Variation In order for selective breeding to be successful, there must be a lot of genetic variation in the population Breeders increase the genetic variation in a population by inducing mutations, which are the ultimate source of genetic variability

Increasing Variation Breeders increase the mutation rate by using radiation and chemicals

Producing New Kinds of Bacteria and Plants Using radiation or chemicals, scientists have been able to develop hundreds of useful bacteria strains Drugs that prevent chromosomal separation during meiosis have been used to produce plants that have many sets of chromosomes. The hybrid plants are polyploid.

What is genetic engineering? In 1973, Mr. Cohen and Mr. Boyer conducted an experiment on the DNA of an American frog.

What is genetic engineering? They found and isolated the gene that codes for ribosomal RNA (rRNA) in the DNA of the frog. They removed that gene from the frog and inserted it into some E. Coli Bacteria.

What Happened? During transcription, the bacteria then produced the frog RNA! Genetic Engineering: the process of manipulating (moving) genes for practical purposes (useful) Recombinant DNA: DNA made from 2 or more organisms that are different.

The Basic Steps of Genetic Engineering Cutting the DNA: 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. Vector: agent used to carry the gene of interest – usually plasmids Plasmid: the circular DNA molecules that replicate

The Basic Steps to Genetic Engineering Making Recombinant DNA DNA fragments of interest (that have already been cut) are combined with the vector. DNA ligase – the enzyme bonds the 2 ends of the fragments to the vectors. Cloning Gene cloning: the process of making many copies of a gene Bacteria reproduce by binary fission

The Basic Steps to Genetic Engineering Screening Cells that have received the gene of interest are separated out. Those cells then continue to produce the protein coded for by the gene

Cutting DNA & Making Recombinant DNA How Restriction enzymes work: The Enzymes recognize specific sequences on Human and Bacterial Plasmids The Enzymes cut the strands. The cut produces DNA fragments with short strands on each end that are complementary to each other “Sticky Ends” Both the human DNA and the Plasmid “Open Up” with the same sticky ends remaining They Bind Together

Recognition sequences Diagram Recognition sequences DNA sequence

Recognition sequences DNA sequence Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end

Confirmation of a Cloned Gene One method used identify a specific gene is called a Southern Blot Steps: Cut DNA from bacteria with restriction enzymes. DNA fragments are separated by a gel soaked in a chemical solution. Gel electrophoresis – uses an electric field within a gel to separate molecules by their size

Confirmation of a Cloned Gene Negatively charged DNA is put into these wells. They are attracted to the positive pole from the electric field. The Smallest DNA fragments move the fastest

Gel Electrophoresis DNA plus restriction enzyme Power source Longer fragments Shorter fragments Mixture of DNA fragments Gel

Confirmation of a Cloned Gene The DNA separated is then transferred to a filter paper (blotted) and a probe solution is added. Probes: radioactive RNA or single-stranded DNA pieces that are complementary to the gene of interest Only DNA fragments complementary to the probe will form and bind bands

Confirmation of Cloned Genes Why do this? Bacterial colonies can be used to produce large quantities of the protein (used to study or make drugs)

Confirmation of Cloned Genes When a bacteria or other cell takes in a foreign piece of DNA such as a plasmid, the process is called transformation If transformation is successful, the recombinant DNA is integrated into one of the chromosomes of the cell.

Improving Crops Genetic engineers can add favorable characteristics to a plant Plants become resistant to insects (no longer need pesticides); resistant to weed killer (so crops won’t die, but weeds will); improved nutrition – rice + corn

Plant Transformation Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome Agrobacterium tumefaciens Gene to be transferred Cellular DNA Recombinant plasmid Plant cell colonies Complete plant is generated from transformed cell Transformed bacteria introduce plasmids into plant cells

Animal Farming Growth hormones is given to cows to produce more milk Human genes are added to farm animals in order to have human proteins in their milk The Human proteins are extracted from milk and sold to pharmacy companies. Useful for complex proteins that can’t be made in bacteria

Creating HGH Human Cell Bacterial Cell Plasmid Gene for human growth hormone Recombinant DNA Gene for human growth hormone Human Cell DNA recombination Sticky ends DNA insertion Bacterial Cell Bacterial chromosome Bacterial cell for containing gene for human growth hormone Plasmid

Animal Farming Transgenic animals: Animals that have foreign DNA in their cells Cloning of animals is another way to make large quantities of a certain protein.

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

Cloning Animals A clone is a member of a population of genetically identical cells produced from a single cell How it works: an intact nucleus from a cell is removed

Cloning Animals The nucleus is fused with a egg cell (whose nucleus has been removed) taken from another adult The fused cell begins to divide and the embryo is placed in the uterus of a foster mother. The “new” egg is then develops normally.

Cloning Embryo A donor cell is taken from a sheep’s udder. Donor Nucleus These two cells are fused using an electric shock. Fused Cell Egg Cell The nucleus of the egg cell is removed. The fused cell begins dividing normally. An egg cell is taken from an adult female sheep. Cloned Lamb The embryo is placed in the uterus of a foster mother. Embryo Foster Mother The embryo develops normally into a lamb—Dolly

Genetically Engineered Drugs and Vaccines Today, many pharmaceutical companies around the world produce important proteins using genetic engineering. Vaccine: a solution containing all or part of a harmless version of a pathogen; used to prevent viral diseases (don’t respond to drugs) Many vaccines are made using genetic engineering

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

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

Three paternity cases tested with DNA purified from blood samples Three paternity cases tested with DNA purified from blood samples. 1: mother; 2: child; 3: alleged father; 4: child + alleged father. M: markers. 3 µg of DNA was loaded per lane. Outcome: A: alleged father excluded; B & C: alleged father confirmed. (Data kindly provided by J. James, Gene Proof Technologies, Nashville, TN, USA.)