Genetic Engineering and Biotechnology IB Biology HL I Spring 2014 Mrs. Peters

What do we already know? What does DNA contain? What does DNA contain? What is the function of a gene? What is the function of a gene? What do you need to make a protein? What do you need to make a protein? How are prokaryotes and eukaryotes different? How are prokaryotes and eukaryotes different?

What is Genetic Engineering? Genetic Engineering: the direct manipulation of DNA for practical purposes used in Forensic Science, agriculture, medicine and food technology used in Forensic Science, agriculture, medicine and food technology Biotechnology: the manipulation (through genetic engineering) of living organisms or their components to produce useful usually commercial products

Important Terms Gene Cloning: making multiple identical copies of a gene (specific pieces of DNA) Gene Cloning: making multiple identical copies of a gene (specific pieces of DNA) Clone: a group of genetically identical organisms or a group of cells derived from a single parent cell. Clone: a group of genetically identical organisms or a group of cells derived from a single parent cell. Plasmid: Plasmid: circular DNA found in bacteria, not part of the nucleoid region Restriction Enzymes: enzymes that protect bacteria by cutting up foreign DNA from invaders Restriction Enzymes: enzymes that protect bacteria by cutting up foreign DNA from invaders DNA ligase: enzyme used to seal DNA strands together DNA ligase: enzyme used to seal DNA strands together

Important Terms Restriction site: the same sequence of 4 to 8 nucleotides, usually symmetrical, where restriction enzymes cut DNA Restriction site: the same sequence of 4 to 8 nucleotides, usually symmetrical, where restriction enzymes cut DNA Restriction Fragment: the piece of DNA that is cut out of a DNA strand by restriction enzymes. Restriction Fragment: the piece of DNA that is cut out of a DNA strand by restriction enzymes. Sticky ends: short single stranded sequences on both sides of a restriction fragment of DNA Sticky ends: short single stranded sequences on both sides of a restriction fragment of DNA Recombinant DNA: a “new” DNA strand which contains the original DNA + a restriction fragment. (recombined DNA) Recombinant DNA: a “new” DNA strand which contains the original DNA + a restriction fragment. (recombined DNA)

Restriction Enzymes Enzymes that cut DNA molecules at specific locations Enzymes that cut DNA molecules at specific locations Discovered in late 1960’s Discovered in late 1960’s Found in Prokaryotes, used naturally to protect bacteria from invading DNA from other organisms Found in Prokaryotes, used naturally to protect bacteria from invading DNA from other organisms Used in science to make DNA fragments and recombinant DNA Used in science to make DNA fragments and recombinant DNA

Making Recombinant DNA 1. Restriction enzymes recognize a specific DNA sequence 2. Restriction enzyme cuts DNA, producing sticky ends 3. DNA fragment from other source is added; fragments stick together by base pairing 4. DNA ligase seals the strands, resulting in recombinant DNA molecule

Time to Practice! RFLP Activity-DNA Scissors RFLP Activity-DNA Scissors

DNA Cloning Process of making multiple identical copies of a gene Process of making multiple identical copies of a gene aka gene cloning aka gene cloning DNA technology uses bacteria plasmids to clone genes DNA technology uses bacteria plasmids to clone genes

DNA and Gene Cloning Process 1. Isolate plasmid DNA from bacteria and DNA gene from other organism 2. Using a restriction enzyme, cut the plasmid and the gene from the other DNA strand 3. Gene is inserted into plasmid and sealed with DNA Ligase, forming recombinant DNA

DNA and Gene Cloning Process 4. Plasmid put into bacterial cell 5. Cell grown in culture, forming clones (lots of copies) 6. Desired gene is identified by products formed (protein or characteristic)

Applications of Cloning Copies of Genes: Copies of Genes: For pest resistance in plants For pest resistance in plants For basic research on genes For basic research on genes To alter bacteria for cleaning toxic waste To alter bacteria for cleaning toxic waste Copies of proteins: Copies of proteins: Human growth hormone treatments Human growth hormone treatments For basic research on proteins For basic research on proteins Dissolving blood clots in heart attack therapy Dissolving blood clots in heart attack therapy

Time to Practice Plasmid Simulation Plasmid Simulation

DNA Profiling DNA profiling is the process of matching DNA from a collected sample to a known individual. DNA profiling is the process of matching DNA from a collected sample to a known individual. DNA samples come from hair, skin, blood and other body fluids DNA samples come from hair, skin, blood and other body fluids DNA profiling is used in forensic science to establish the possibility of guilt or prove a suspect innocent; also used in paternity testing DNA profiling is used in forensic science to establish the possibility of guilt or prove a suspect innocent; also used in paternity testing What happens on CSI and NCIS What happens on CSI and NCIS

PCR Polymerase Chain Reaction Polymerase Chain Reaction a technique in which a specific piece of DNA is copied quickly without the use of cells a technique in which a specific piece of DNA is copied quickly without the use of cells Used for DNA profiling in forensic science and paternity testing Used for DNA profiling in forensic science and paternity testing

PCR: What is the machine doing? PCR Process: PCR Process: DNA is incubated in a test tube with DNA polymerase, nucleotides and short pieces of synthetic single-stranded DNA to act as primers for DNA synthesis DNA is incubated in a test tube with DNA polymerase, nucleotides and short pieces of synthetic single-stranded DNA to act as primers for DNA synthesis This is a three-cycle process which continues until the targeted sequence has been duplicated many times. This is a three-cycle process which continues until the targeted sequence has been duplicated many times.

PCR: What is the machine doing? PCR Process:  Heat briefly to separate DNA strands  Cool to allow primers to hydrogen bond  DNA polymerase adds nucleotides to the 3’ end of each primer  After 1 cycle, 2 DNA molecules are made  Process repeats, each cycle takes about 5 minutes and doubles the targeted DNA sequence each time.

PCR: What is the machine doing? PCR End Result: millions of identical DNA fragments These copies are used to produce a DNA profile which can be used in crime scene analysis and paternity testing.

DNA Analysis Gel electrophoresis Gel electrophoresis Technique used to separate macromolecules based on size or electrical charge Technique used to separate macromolecules based on size or electrical charge Sorts DNA fragments by size in bands containing molecules of the same length Sorts DNA fragments by size in bands containing molecules of the same length Restriction fragment analysis detects DNA differences Restriction fragment analysis detects DNA differences Use restriction enzymes to cut DNA and electrophoresis to separate fragments Use restriction enzymes to cut DNA and electrophoresis to separate fragments

Gel Electrophoresis Process 1. Samples are placed in wells at one end of the gel 2. Electrodes are attached to electrophoresis chamber 3. Molecules migrate toward the opposite pole, depending on charge 4. When current is turned off, molecules are arrayed in bands along a lane according to size

Uses of Electrophoresis DNA Profiling for DNA Profiling for Paternity Tests Paternity Tests Forensic investigation Forensic investigation DNA Analysis DNA Analysis Human Genome Project Human Genome Project

Time to Practice! Electrophoresis Simulation: DNA Goes to the Races Electrophoresis Simulation: DNA Goes to the Races

Human Genome Project In 1990, the Human Genome Project started as an international collaboration to determine the entire base sequence of the human genome. In 1990, the Human Genome Project started as an international collaboration to determine the entire base sequence of the human genome. Public research groups (universities) and private companies (Celera) worked on the project Public research groups (universities) and private companies (Celera) worked on the project Completed in 2003 Completed in 2003 Continue to work on locating genes and mapping on specific chromosomes Continue to work on locating genes and mapping on specific chromosomes

Human Genome Project Outcomes: Value in knowing the sequence of genes on chromosomes, useful in medicine, forensics and evolution Value in knowing the sequence of genes on chromosomes, useful in medicine, forensics and evolution Medical benefits: Medical benefits: Improved diagnosis of disease Improved diagnosis of disease Early detection of genetic susceptibility to disease Early detection of genetic susceptibility to disease Better identification of carriers of genetic disorders Better identification of carriers of genetic disorders Drug design to find new classes of drugs to act on specific genes Drug design to find new classes of drugs to act on specific genes

Human Genome Project Outcomes: Improved techniques have made it possible to find genomes of other organisms Improved techniques have made it possible to find genomes of other organisms Provided new insights into role of “junk DNA” (sections of DNA that are not transcribed) Provided new insights into role of “junk DNA” (sections of DNA that are not transcribed) Found far fewer protein coding regions than expected Found far fewer protein coding regions than expected Humans have about 25,000 genes Humans have about 25,000 genes

Human Genome Project Outcomes cont’d Knowledge of the significance of certain sequences Knowledge of the significance of certain sequences Presence or absence can be detected using microarray technology Presence or absence can be detected using microarray technology New research fields have emerged New research fields have emerged Bioinformatics: use of computers to store and analyze huge amounts of data being generated by sequencing the genome Bioinformatics: use of computers to store and analyze huge amounts of data being generated by sequencing the genome Pharmacogenomics: links differences in genomic information in different populations to differences in their response to drug treatment Pharmacogenomics: links differences in genomic information in different populations to differences in their response to drug treatment

Cloning Eukaryotic Cells  A cell is taken from the desired organism; an egg cell is taken from a surrogate  The nucleus is removed from the egg  The desired cell and the nucleus free egg are fused together  The egg grows to become an embryo  Embryo is implanted in the surrogate and develops into the desired organism

GMOs Genetically Modified Organisms: organisms in which their genetic make up has been altered Genetically Modified Organisms: organisms in which their genetic make up has been altered Benefits to GM products: Benefits to GM products: Crops: enhanced taste and quality; reduced maturation time; increased nutrients, yields and stress tolerance; improved resistance to disease, pests and herbicides Crops: enhanced taste and quality; reduced maturation time; increased nutrients, yields and stress tolerance; improved resistance to disease, pests and herbicides Animals: increased resistance, productivity, hardiness, and feed efficiency; better yields of meat, eggs, and milk; improved animal health Animals: increased resistance, productivity, hardiness, and feed efficiency; better yields of meat, eggs, and milk; improved animal health Environment: “friendly” bioherbicides and bioinsecticides; conservation of soil, water, and energy; better natural waste management; more efficient processing Environment: “friendly” bioherbicides and bioinsecticides; conservation of soil, water, and energy; better natural waste management; more efficient processing Society: increased food security for growing populations Society: increased food security for growing populations

GMOs Controversies: Controversies: Safety: potential human health impact (allergies, transfer of antibiotic resistance), unknown potential environmental impact, unknown effect on other organisms, loss of flora and fauna biodiversity Safety: potential human health impact (allergies, transfer of antibiotic resistance), unknown potential environmental impact, unknown effect on other organisms, loss of flora and fauna biodiversity Access and Intellectual Property: domination of world food production by few companies; increased dependence on industrial nations by developing countries; Access and Intellectual Property: domination of world food production by few companies; increased dependence on industrial nations by developing countries; Ethics: violation of natural organisms’ intrinsic values; tampering with nature by mixing genes among species; objections to consuming animal genes in plants and vice versa; stress for animals Ethics: violation of natural organisms’ intrinsic values; tampering with nature by mixing genes among species; objections to consuming animal genes in plants and vice versa; stress for animals Labeling: not mandated by some countries (US); mixing GM crops with non-GM crops Labeling: not mandated by some countries (US); mixing GM crops with non-GM crops Society: new advances may be skewed to interest rich countries Society: new advances may be skewed to interest rich countries

GMOs Current Examples Current Examples *Salt tolerance in tomato plant *Salt tolerance in tomato plant *Synthesis of beta-carotene in rice (golden rice) *Synthesis of beta-carotene in rice (golden rice) *Factor IX (for human blood clotting) in sheep milk *Factor IX (for human blood clotting) in sheep milk Herbicide resistance in crop plants (round-up ready plants) Herbicide resistance in crop plants (round-up ready plants) Glowing Bacteria… you will do this! Glowing Bacteria… you will do this! (* = Examples from IB, must know 2)

Issues in Biotechnology Benefits and harmful effects of genetic modification Benefits and harmful effects of genetic modification Ethical Issues of therapeutic cloning in humans: creating an embryo to supply embryonic stem cells for medical use Ethical Issues of therapeutic cloning in humans: creating an embryo to supply embryonic stem cells for medical use need more here

Cloning Eukaryotic Cells