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Topic 3: Genetics Topic 3. 5 Genetic Modification and Biotechnology Topic 3: Genetics Topic 3. 5 Genetic Modification and Biotechnology

Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

Ancient biotech: Selective breeding Mating organisms to produce offspring with desired traits (has been Ancient biotech: Selective breeding Mating organisms to produce offspring with desired traits (has been around since ancient times) Inbreeding= mating between closely related individuals Hybrids– offspring of parents with different forms of traits. In plants, hybrids are often stronger or bigger.

Modern Techniques in Biotech… Modern Techniques in Biotech…

Genetic Engineering/ Genetic Modification: The transfer of genes between different species (i. e. cutting Genetic Engineering/ Genetic Modification: The transfer of genes between different species (i. e. cutting out DNA from one organism and putting it into another. ) – makes recombinant DNA Recombinant DNA: DNA in which genes from 2 different organisms are linked Transgenic organism= an organism with recombinant DNA C: Documents and SettingsBBAUGHMANDesktopCOURSESAP BIOarticles C: Documents and SettingsBBAUGHMANDesktopCOURSESLSSI Summer Institute ResourcesLSSI Prog. ResourcesGFP Mice

Genetic Engineering Tools Restriction Enzymes DNA ligase Vectors – Plasmids! – Viruses Reverse Transcriptase Genetic Engineering Tools Restriction Enzymes DNA ligase Vectors – Plasmids! – Viruses Reverse Transcriptase

Restriction Endonucleases aka: Restriction enzymes : – They cut up the DNA (restriction) at Restriction Endonucleases aka: Restriction enzymes : – They cut up the DNA (restriction) at very specific sequences – in nature, these enzymes protect bacteria from intruding DNA – Restriction site: where the restriction enzyme cuts the DNA (a specific sequence) – Sticky end: short single-stranded extensions of restriction fragments (segments of DNA cut by restriction enzymes in a reproducible way)

Restriction Enzymes http: //highered. mcgrawhill. com/sites/0072437316/student_view 0/chapter 16/animations. html# C: Documents and SettingsBBAUGHMANDesktopbio powerpointsChapter Restriction Enzymes http: //highered. mcgrawhill. com/sites/0072437316/student_view 0/chapter 16/animations. html# C: Documents and SettingsBBAUGHMANDesktopbio powerpointsChapter 13 BDOL IC

DNA ligase: enzyme that joins the DNA fragments covalently (called gene splicing) Vector: Something DNA ligase: enzyme that joins the DNA fragments covalently (called gene splicing) Vector: Something that carries foreign DNA into a cell. – Bacterial plasmids… small circles of DNA – viruses

Restriction Enzymes and Ligase https: //www. dnalc. org/view/15476 -Mechanism-of-Recombination -3 D-animation-with-basic-narration. html Restriction Enzymes and Ligase https: //www. dnalc. org/view/15476 -Mechanism-of-Recombination -3 D-animation-with-basic-narration. html

Bacterial plasmids in gene cloning Bacterial plasmids in gene cloning

Gene Cloning Clone = genetically identical copy Gene cloning: Making more copies of a Gene Cloning Clone = genetically identical copy Gene cloning: Making more copies of a gene – Bacterial plasmid with recombinant DNA reproduces as the bacteria reproduce. – C: Documents and SettingsBBAUGHMANDesktopbio powerpointsChapter 13 BDOL IC

Summary of Steps for gene cloning Isolation of cloning vector (bacterial plasmid) & DNA Summary of Steps for gene cloning Isolation of cloning vector (bacterial plasmid) & DNA (gene of interest) Insertion of gene of interest into the cloning vector (how? ) Introduction of cloning vector into cells (transformation of bacterial cells) Cloning of cells (and foreign genes) Identification of cell clones carrying the gene of interest – (nucleic acid hybridization– use radioactively labeled nucleic acid probe) or – look for protein product http: //highered. mcgrawhill. com/sites/0072437316/student_view 0/c hapter 16/animations. html#

Application: Universality of the Genetic Code One key example: Production of human insulin in Application: Universality of the Genetic Code One key example: Production of human insulin in bacteria

Examples of GMO’s Beta-carotene rice (a. k. a. Golden rice, vitamin A rice)-daffodil and Examples of GMO’s Beta-carotene rice (a. k. a. Golden rice, vitamin A rice)-daffodil and soil bacterium genes Bt corn and other crops— Bacillus thuringiensis gene. Terminator technologies– cause sterility of seeds.

Application and Skills: Application: Assessment of the potential risks and benefits associated with genetic Application and Skills: Application: Assessment of the potential risks and benefits associated with genetic modification of crops. Read pages 192 -195 at home tonight and take summary notes on the most salient points. Skill: Analysis of data on risks to monarch butterflies of Bt crops. (see p. 194 -195 and answer Data-based questions: Transgenic pollen and monarch larvae in your assignments notebook) Watch “Future of Food” here.

Cloning • Clones= genetically identical organisms, derived from a single parent cell. • Many Cloning • Clones= genetically identical organisms, derived from a single parent cell. • Many plant species and some animal species have natural methods of cloning.

Skill: Investigating Cloning in Plants Design of an experiment to assess one factor affecting Skill: Investigating Cloning in Plants Design of an experiment to assess one factor affecting the rooting of stem-cuttings. (see page 197 for ideas) Note: possible inspiration for IA.

Animal Cloning Methods • 1. Breaking embryo up in to more than one group Animal Cloning Methods • 1. Breaking embryo up in to more than one group of cells (how identical twins form) • 2. Cloning adult animals with differentiated cells • ex. Dolly the sheep • Application: Somatic cell nuclear transfer • Remove Nucleus from an egg • inject somatic cell nucleus into egg OR… • Fuse this unfertilized egg with a body cell • http: //www. youtube. com/watch? v =bb. Zi. Oi. PVG 6 c Somatic cell nuclear transfer

 • Ethical issues (discussion) • Reproductive cloning • Therapeutic cloning– creation of an • Ethical issues (discussion) • Reproductive cloning • Therapeutic cloning– creation of an embryo to provide stem cells

DNA Analysis & Genomics PCR (polymerase chain reaction) Gel electrophoresis Restriction fragment analysis (RFLPs) DNA Analysis & Genomics PCR (polymerase chain reaction) Gel electrophoresis Restriction fragment analysis (RFLPs) DNA sequencing Human genome project

Polymerase chain reaction (PCR) Replication of DNA without cells (in vitro) Used to amplify Polymerase chain reaction (PCR) Replication of DNA without cells (in vitro) Used to amplify small amount of DNA Materials in tube: primers (single-stranded DNA), Taq polymerase, nucleotides Heat and cool in thermal cycler Applications: fossils, forensics, prenatal diagnosis, etc. http: //highered. mcgrawhill. com/sites/0072437316/student_view 0/chapter 16

DNA Analysis Gel electrophoresis: separates nucleic acid fragments on the basis of size creating DNA Analysis Gel electrophoresis: separates nucleic acid fragments on the basis of size creating bands of DNA consisting of pieces of the same length. Used in DNA Profiling – Paternity tests – Forensics Ethical issues? Good DNA ANALYSIS COMPUTER LAB ACTIVITY= http: //www. biology. arizona. edu/human_bio/activities/blackett/introduction. html See Activities for in class simulation to be used forensics and paternity tests

How does gel electrophoresis work? DNA is cut with restriction enzymes and added to How does gel electrophoresis work? DNA is cut with restriction enzymes and added to a well. The negative DNA is attracted to the positive electrode. smaller pieces of DNA move faster through the pores in the gel A unique banding pattern is observed. The human genome is estimated to be about 3 billion base pairs long and to contain 20, 000 -25, 000 distinct genes

Gel Electrophoresis animationhttp: //www. sumanasinc. com/webcontent/anisamples/majorsbiology/g elelectrophoresis. html http: //www. dnalc. org/ddnalc/resources/animations. html http: Gel Electrophoresis animationhttp: //www. sumanasinc. com/webcontent/anisamples/majorsbiology/g elelectrophoresis. html http: //www. dnalc. org/ddnalc/resources/animations. html http: //learn. genetics. utah. edu/units/biotech/gel/ good gel electrophoresis virtual lab

DNA Analysis (continued) Restriction fragment length polymorphisms (RFLPs)= differences between different individuals in lengths DNA Analysis (continued) Restriction fragment length polymorphisms (RFLPs)= differences between different individuals in lengths of restriction fragments – Found especially in satellite DNA (highly repetitive non-coding sequences of DNA) RFLP animation http: //highered. mcgrawhill. com/sites/0072437316/student_view 0/chapter 16/animations. html#

Skill: Analysis of examples of DNA profiles. LANES - 1 2 3 4 Injection Skill: Analysis of examples of DNA profiles. LANES - 1 2 3 4 Injection wells DNA + Based on what you just learned, which lane contains the largest fragment of DNA? The shortest? What is the fancy 4 word term used to describe the fact that people have different size restriction fragments?

Skill: Analysis of examples of DNA profiles. Use the DNA gel, and your knowledge Skill: Analysis of examples of DNA profiles. Use the DNA gel, and your knowledge of DNA fingerprint to analyze and determine which suspect’s DNA matches the DNA at the crime scene. DNA at crime Suspect scene 1 2 3 - +

Skill: Analysis of examples of DNA profiles. (Paternity Problem) • Look at the electrophoresis Skill: Analysis of examples of DNA profiles. (Paternity Problem) • Look at the electrophoresis gel picture on the right • Two of the individuals are parents and one is their child. • Answer the following: • Which individual is the child? • How many bands of DNA does the child share with the mother? The father? Explain why. • Based off of the gel, what color are the mother’s eyes? • Just kidding…

O. J. Simpson capital murder case, 1/95 -9/95 Odds of blood in Ford Bronco O. J. Simpson capital murder case, 1/95 -9/95 Odds of blood in Ford Bronco not being R. Goldman’s: 6. 5 billion to 1 Odds of blood on socks in bedroom not being N. Brown. Simpson’s: 8. 5 billion to 1 Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O. J. Simpson: 21. 5 billion to 1 Number of people on planet earth: 6. 1 billion Odds of being struck by lightning in the U. S. : 2. 8 million to 1 Odds of winning the Illinois Big Game lottery: 76 million to 1 Odds of getting killed driving to the gas station to buy a lottery ticket 4. 5 million to 1 Odds of seeing 3 albino deer at the same time: 85 million to 1 Odds of having quintuplets: 85 million to 1 Odds of being struck by a meteorite: 10 trillion to 1

Theory of knowledge: Discuss • The use of DNA for securing convictions in legal Theory of knowledge: Discuss • The use of DNA for securing convictions in legal cases is well established, yet even universally accepted theories are overturned in the light of new evidence in science. What criteria are necessary for assessing the reliability of evidence?

DNA Sequencing Determination of nucleotide sequences Genomics: the study of genomes Genome: the whole DNA Sequencing Determination of nucleotide sequences Genomics: the study of genomes Genome: the whole of the genetic information of an organism. The Human Genome Project sequenced the entire base sequence of human DNA. Human Genome Project Animations: http: //www. genome. gov/250 19885

Application: Use of nucleotides containing dideoxyribose to stop DNA replication in preparation of samples Application: Use of nucleotides containing dideoxyribose to stop DNA replication in preparation of samples for base sequencing. – Dideoxyribonucleotides with different nitrogen bases are tagged with different colored fluorescent markers. – Since the 3’ OH is missing, condensation can’t continue – So reaction stops. – Electrophoresis can be used to separate by size and determine sequence.

Human Genome Project Outcomes: – Identification and sequencing of genes involved in traits and Human Genome Project Outcomes: – Identification and sequencing of genes involved in traits and disease. – Genetic testing improved. – Greater understanding of genetics. – Ethical issues with genetic testing etc. http: //www. pbs. org/wgbh/nova/scie ncenow/archive/title-m-z. html Public Genomes or Personal genetic Testing

International-mindedness Sequencing of the human genome shows that all humans share the vast majority International-mindedness Sequencing of the human genome shows that all humans share the vast majority of their base sequences but also that there are many single nucleotide polymorphisms that contribute to human diversity.

Application: Comparison of the number of genes in humans with other species. (adapted from Application: Comparison of the number of genes in humans with other species. (adapted from http: //ibworld. me/Genetics. php) Species (#genes) Reference human ~21, 000 ref E. coli K-12 4, 377 http: //users. rcn. com/jkimball. ma. ultranet/Biology. Pages/G /Genome. Sizes. html rice 46, 022 to 55, 615 ref dog ~25, 000 ref fruit fly 13, 600 ref mouse ~23, 000 ref Guidance: The number of genes in a species should not be referred to as genome size as this term is used for the total amount of DNA. At least one plant and one bacterium should be included in the comparison and at least one species with more genes and one with fewer genes than a human.

International-mindedness Sequencing of the rice genome involved cooperation between biologists in 10 countries. International-mindedness Sequencing of the rice genome involved cooperation between biologists in 10 countries.

Skill: Assignment as a Class Use of a database to determine differences in the Skill: Assignment as a Class Use of a database to determine differences in the base sequence of a gene in two species. Here is the link to the Gen. Bank site: http: //www. ncbi. nlm. nih. gov/pubmed/ Choose “Gene” from the search menu. Enter the name of a gene plus an organism and click “Search. ” Ex. “cytochrome oxidase 1 for homo sapiens” Click on the name of the gene that has the correct description. Once loaded, scroll down to where it says “Genomic regions, transcripts, and products” and click on “FASTA” Copy the entire sequence beginning with where it says “>gi” Paste this into a text file (Notepad or Wordpad) Repeat with all species that you want to compare and save the files Download Clustal. X and run it. (http: //www. clustal. org/download/current/ ) File “Load Sequences” and choose your first sequence File “Append Sequences” and choose your next sequence… Under alignment menu choose “Do Complete Alignment”

Assignment at home Use a database to determine differences in the base sequence of Assignment at home Use a database to determine differences in the base sequence of a gene in two species. Submit your response to the Google Form Below: https: //docs. google. com/forms/d/e/1 FAIp. QLScw 6 ZSh. HWGKep. XSn. SJdww. Yv. Uv 4 ujwa-H 1 s. A 0 Iymley. ZZHIWa. Q/viewform? usp=send_form

Application: Comparison of genome size in T 2 phage, Escherichia coli, Drosophila melanogaster, Homo Application: Comparison of genome size in T 2 phage, Escherichia coli, Drosophila melanogaster, Homo sapiens and Paris japonica. Organism Genome Size (million base pairs) Description T 2 phage 0. 18 Virus that attacks E. coli 5 Gut bacterium Drosophila melanogaster 140 Fruit Fly Homo sapiens 3000 Humans Paris japonica 150, 000 Woodland Plant

Extra slides… cut from presentation Extra slides… cut from presentation

Practical DNA Technology Uses Diagnosis of disease Human gene therapy Pharmaceutical products (vaccines) http: Practical DNA Technology Uses Diagnosis of disease Human gene therapy Pharmaceutical products (vaccines) http: //highered. mcgrawhill. com/sites/0072437316/student_view 0/chapter 16/anima tions. html# Forensics Animal husbandry (transgenic organisms) Genetic engineering in plants Ethical concerns?

Skip: Option F: Reverse Transcriptase Catalyses the production of DNA from RNA Why is Skip: Option F: Reverse Transcriptase Catalyses the production of DNA from RNA Why is this helpful in making recombinant DNA? – Hint: Think about introns and exons… – Ex. Bacteria that make human insulin…

Skip: Option F: RNA viruses Where does Reverse Transcriptase come from? Answer: Retroviruses: transcribe Skip: Option F: RNA viruses Where does Reverse Transcriptase come from? Answer: Retroviruses: transcribe DNA from an RNA template (RNA--->DNA) – DNA gets incorporated as provirus in host Ex. HIV http: //highered. mcgrawhill. com/sites/0072437316/student_view 0/chapter 26/animations. html http: //www. pbs. org/wgbh/nova/aids/action. h

Skip (option f) Gene Therapy Germ line Therapy: reproductive cells– affects offspring Somatic Therapy: Skip (option f) Gene Therapy Germ line Therapy: reproductive cells– affects offspring Somatic Therapy: body cells of an individual Ex. White blood cells of SCID (severe combined immunodeficiency) 1. Blood cells are removed 2. Engineer viral DNA 3. Use of viral vector 4. Blood cells replaced, having needed gene. Risks?