DNA Technology In laboratory experiments Genes can be transcribed and translated after being transplanted from one species to another Called “Recombinant DNA” technology Can be produced via “Genetic Engineering” (laboratory manipulation)

Overview: Understanding and Manipulating Genomes One of the greatest achievements of modern science has been the sequencing of the human genome, which was largely completed by 2003 DNA sequencing accomplishments Have all depended on advances in DNA technology, starting with the invention of methods for making recombinant DNA DNA sequencing animation

How can we modify a person’s genome? Genomics How can we modify a person’s genome? Gene therapy - insertion of genetic material into human cells to treat a disorder Ex vivo therapy – cells are removed for a person altered and then returned to the patient In vivo therapy – a gene is directly inserted into an individual through a vector (e.g. viruses) or directly injected to replace mutated genes or to restore normal controls over gene activity Gene therapy has been most successful in treating cancer, to date.

Ex vivo gene therapy Genomics 2. Use retroviruses 1. Remove bone defective gene retrovirus normal gene viral recombinant RNA viral recombinant DNA 1. Remove bone marrow stem cells. 2. Use retroviruses to bring the normal gene into the bone 4. Return genetically engineered cells to patient. 3. Viral recombinant DNA carries normal gene into genome. viral recombinant RNA reverse transcription Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

DNA Cloning Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment To work directly with specific genes Scientists have developed methods for preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning

24.3 DNA Technology The Cloning of a Gene Cloning: Production of many identical copies of an organism through some asexual means. Gene Cloning: The production of many identical copies of a single gene Two Ways to Clone a Gene: Recombinant DNA Polymerase Chain Reaction

Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites

Restriction Enzymes and Sticky Ends Step through animation of cut/splice using EcoRI Narrated animation

Cloning of a Human Gene / Recombinant DNA Restriction enzymes breaks open a plasmid vector at specific sequence of bases “sticky ends” Foreign DNA that is to be inserted is also cleaved with same restriction enzyme so ends match Foreign DNA is inserted into plasmid DNA and “sticky ends” pair up DNA ligase seals them together Narrated animation of “Cloning a Gene”

24.3 DNA Technology Polymerase Chain Reaction Amplifies a targeted DNA sequence Requires DNA polymerase, a set of primers, and a supply of nucleotides Primers are single stranded DNA sequences that start replication process Amount of DNA doubles with each replication cycle Process is now automated Narrated animation Step by step animation

24.3 DNA Technology DNA Fingerprinting Permits identification of individuals and their relatives Based on, polymorphisms differences between sequences in nucleotides between individuals RFLPs : restriction fragment length polymorphisms Narrated animation Detection of the number of repeating segments (called repeats) are present at specific locations in DNA Different numbers in different people PCR amplifies only particular portions of the DNA Procedure is performed at several locations to identify repeats

DNA Fingerprints DNA fragments (after digest with restriction enzymes) can be separated through gel ELECTROPHORESIS See How: Animation http://highered.mcgraw-hill.com/sites/007337797x/student_view0/chapter14/animation_quiz_-_dna_fingerprinting.html Step-by-step electrophoresis Another walk-through explanation

Forensic Evidence DNA “fingerprints” obtained by analysis of tissue or body fluids found at crime scenes Can provide definitive evidence that a suspect is guilty or, more specifically, not guilty Is a specific pattern of bands of RFLP markers on a gel Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V) D Jeans shirt V 4 g 8 g Figure 20.17

DNA fingerprinting Can also be used in establishing paternity Figure: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint. http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Paternity_testing

24.3 DNA Technology Biotechnology Biotechnology uses natural biological systems to create a product or to achieve a goal desired by humans. “Model Organisms” favored for genetics research

Environmental Cleanup Genetic engineering can be used to modify the metabolism of microorganisms So that they can be used to extract minerals from the environment or degrade various types of potentially toxic waste materials

24.3 DNA Technology Transgenic Bacteria Medical Uses: Production of Insulin, Human Growth Hormone, Hepatitis B Vaccine Agricultural Uses: Bacteria that protects plants from freezing, bacteria that protect plant roots from insects Environmental: Bacteria that degrade oil (clean up after oil spills), bacteria that remove sulfur from coal

Transgenic (GM) Plants 24.3 DNA Technology Transgenic (GM) Plants Plants have been engineered to secrete a toxin that kills insects (ex: Bt corn) Plants have been engineered to be resistant to herbicides (ex: Roundup Ready) Animation: Gene Transfer in Plants Using a Ti Plasmid http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter17/genes_into_plants_using_the_ti-plasmid.html

Agricultural Applications DNA technology Is being used to improve agricultural productivity and food quality In 2008: 92% of the soybeans and 80% of the corn planted in the United States had been genetically engineered.

Genetic Engineering in Plants Agricultural scientists Have already endowed a number of crop plants with genes for desirable traits Bt corn (right)

Biotechnology products: Transgenic plants DNA technology Biotechnology products: Transgenic plants Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transgenic Crops of the Future Improved Agricultural Traits Disease-protected Wheat, corn, potatoes Herbicide-resistant Wheat, rice, sugar beets, canola Salt-tolerant Cereals, rice, sugarcane Drought-tolerant Cereals, rice, sugarcane Cold-tolerant Cereals, rice, sugarcane Improved yield Cereals, rice, corn, cotton Modified wood pulp Trees Improved Food Quality Traits Fatty acid/oil content Corn, soybeans Protein/starch content Cereals, potatoes, soybeans, rice, corn Amino acid content Corn, soybeans a. Desirable traits b. Salt-intolerant Salt-tolerant

Health focus: Ecological concern about GMO crops DNA technology Health focus: Ecological concern about GMO crops Resistance increasing in the target pest Exchange of genetic material between the transgenic plant and a related species Concern about the impact of BT crops on nontarget species (ex: pollinators)

GM Animals and “Pharm” Animals Transgenic animals Contain genes from other organisms Sometimes called “chimeras” Fig 1. transgenic mouse lines expressing GFP known as “green mice.”

24.3 DNA Technology Transgenic Animals Fish, cows, pigs, rabbits and sheep have been engineered to produce human growth hormone in order to increase size of the animals

Transgenic organisms have a foreign gene inserted into their DNA Have been engineered to be pharmaceutical “factories” “Pharm” Animals Figure 20.18 Human breast milk from a cow (2011)

Pharmaceutical Products

http://www.nca-nl.org/English/Newsletters/Nb13/nl13txt.html “Knockout” mice A number of mice models have been developed: either possessing an inactivated tumor suppressor gene (p53), an activated oncogene (Tg.AC), over-expression of a (human) oncogene (rasH2) or being deficient in nucleotide excision repair (Xpa, de Vries et al., 1995). These mice models have several advantages: the number of animals needed for one study is 120 instead of 400-500 the duration of the study is 6-9 instead of 24 months leading to less distress of the animals the transgenic mouse model is considered more discriminating hence improving the accuracy and reliability of human carcinogen identification.

Safety and Ethical Questions Raised by DNA Technology The potential benefits of genetic engineering Must be carefully weighed against the potential hazards of creating products or developing procedures that are harmful to humans or the environment Today, much public concern about possible hazards Centers on genetically modified (GMOs) organisms used as food (allergic reactions, etc) Gene “escape”