GENETIC ENGINEERING and BIOTECHNOLOGY Topic 4.4 IB Biology Miss Werba

TOPIC 4 - GENETICS 4.1 CHROMOSOMES, ALLELES, GENES & MUTATIONS 4.4 4.2 MEIOSIS 4.3 THEORETICAL GENETICS 4.4 GENETIC ENGINEERING & BIOTECHNOLOGY J WERBA – IB BIOLOGY 2

THINGS TO COVER Polymerase Chain Reaction (PCR) Gel electrophoresis DNA profiling Paternity and forensic investigations Human Genome Project Gene transfer Genetically modified (GM) crops & animals Cloning Ethics of cloning J WERBA – IB BIOLOGY 3

BEFORE WE BEGIN.... TOOLS OF THE TRADE

TOOLS OF THE TRADE: RESTRICTION ENZYMES 4.4.8 TOOLS OF THE TRADE: RESTRICTION ENZYMES Enzymes which recognise a specific repeating base sequence of DNA They cut at a specific point in this sequence Create “sticky ends” with known DNA sequences that can be utilised for genetic engineering eg. HindIII, EcoR1 MISS J WERBA - TERM 2 2011 5

TOOLS OF THE TRADE: RESTRICTION ENZYMES 4.4.8 TOOLS OF THE TRADE: RESTRICTION ENZYMES MISS J WERBA - TERM 2 2011 6

TOOLS OF THE TRADE: REVERSE TRANSCRIPTASE 4.4.8 TOOLS OF THE TRADE: REVERSE TRANSCRIPTASE It reverses transcription! Enzymes which can cause a piece of RNA to produce a matching piece of DNA The DNA can then be made double-stranded and used to transfer a genetic characteristic to another organism DNA made is called copy DNA (cDNA) MISS J WERBA - TERM 2 2011 7

TOOLS OF THE TRADE: REVERSE TRANSCRIPTASE 4.4.8 TOOLS OF THE TRADE: REVERSE TRANSCRIPTASE MISS J WERBA - TERM 2 2011 8

TOOLS OF THE TRADE: DNA POLYMERASE 4.4.8 TOOLS OF THE TRADE: DNA POLYMERASE Enzyme used for making multiple copies of DNA Enzyme allows single-stranded cDNA to be made double-stranded Also allows small DNA samples to be replicated so that they can be tested multiple times if necessary eg. DNA from an embryo during aminocentesis, DNA from a crime scene MISS J WERBA - TERM 2 2011 9

TOOLS OF THE TRADE: DNA LIGASE 4.4.8 TOOLS OF THE TRADE: DNA LIGASE Enzyme used for joining pieces of DNA which are being spliced together ie. genetic “glue” Allows an isolated or created piece of DNA to be joined to the DNA of a recipient organism. MISS J WERBA - TERM 2 2011 10

TOOLS OF THE TRADE: GENE PROBES 4.4.8 TOOLS OF THE TRADE: GENE PROBES Single-stranded DNA used to identify genes with base sequences complementary to the probe. Probes are made using reverse transcriptase enzymes. Probes are radioactively labelled so that where the probe pairs with the gene, the probe and the gene can be identified. MISS J WERBA - TERM 2 2011 11

TOOLS OF THE TRADE: MARKER GENES 4.4.8 TOOLS OF THE TRADE: MARKER GENES Genes that are close to the gene being studied that produce an obvious phenotype so it can be easily identified. Presence of the marker indicates that the neighbouring gene has probably been inherited. In genetic engineering, a gene is normally introduced into an organism together with its marker. MISS J WERBA - TERM 2 2011 12

4.4.8 TOOLS OF THE TRADE MISS J WERBA - TERM 2 2011 13

GENETIC ENGINEERING and BIOTECHNOLOGY AND NOW.... GENETIC ENGINEERING and BIOTECHNOLOGY

POLYMERASE CHAIN REACTION (PCR) 4.4.1 POLYMERASE CHAIN REACTION (PCR) Polymerase Chain Reaction (PCR) is a way of producing large quantities of a specific target sequence of DNA It is useful when only a small amount of DNA is available for testing eg. crime scene samples of blood, semen, tissue, hair, etc. J WERBA – IB BIOLOGY 15

POLYMERASE CHAIN REACTION (PCR) 4.4.1 POLYMERASE CHAIN REACTION (PCR) PCR involves a repeated procedure of 3 steps: Denaturation: DNA is heated to separate it into 2 strands Annealing: DNA primers attach to opposite ends of the target sequence Elongation: DNA polymerase copies the strands One cycle of PCR yields two identical copies of the DNA sequence A standard reaction of 30 cycles would yield over a million copies of the DNA J WERBA – IB BIOLOGY 16

POLYMERASE CHAIN REACTION (PCR) 4.4.1 POLYMERASE CHAIN REACTION (PCR) J WERBA – IB BIOLOGY 17

PCR

GEL ELECTROPHORESIS Command term = STATE 4.4.2 GEL ELECTROPHORESIS Command term = STATE In gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size. Process: DNA fragmented with restriction enzymes Loaded into a gel and an electric current is applied DNA moves towards the anode (+ end) The fragments are separated according to their size small fragments move faster, separate further J WERBA – IB BIOLOGY 19

GEL ELECTROPHORESIS Command term = STATE 4.4.2 GEL ELECTROPHORESIS Command term = STATE J WERBA – IB BIOLOGY 20

DNA PROFILING Command term = STATE 4.4.3 DNA PROFILING Command term = STATE Gel electrophoresis is used in DNA profiling. J WERBA – IB BIOLOGY 21

DNA PROFILING Command term = STATE 4.4.3 DNA PROFILING Command term = STATE DNA profiling is a technique by which individuals are identified on the basis of their DNA profiles. Non-coding regions of an individual's genome contain satellite DNA - long stretches of DNA made up of repeating elements called short tandem repeats (STRs). These repeating sequences can be excised to form fragments, by cutting with a variety of restriction enzymes. As individuals all have a different number of repeats, they will all generate unique fragment profiles. These different profiles can be compared using gel electrophoresis. J WERBA – IB BIOLOGY 22

4.4.4 USES OF DNA PROFILING The gel electrophoresis identifies an individual’s banding pattern Restriction enzymes (endonucleases) target non-coding elements (STRs) that differ in length in individuals It can be used to determine paternity and also be used in forensic investigations J WERBA – IB BIOLOGY 23

USES OF DNA PROFILING Is the man the child’s father? 4.4.5 USES OF DNA PROFILING Is the man the child’s father? Yes. The DNA sequence of the child can be mapped to show that similar bands exist in the father's sequence which are not present in the mother's sequence. J WERBA – IB BIOLOGY 24

THE HUMAN GENOME PROJECT 4.4.6 THE HUMAN GENOME PROJECT The Human Genome Project was an international cooperative venture to sequence the complete human genome. Humans have about 30,000-40,000 genes. It was started in 1990 and was finished in 2003. Y chromosome X chromosome J WERBA – IB BIOLOGY 25

OUTCOMES OF THE HUMAN GENOME PROJECT 4.4.6 OUTCOMES OF THE HUMAN GENOME PROJECT Mapping (locating genes) Screening (for disease) Treatment (gene therapy & targeted drug design) eg. It will allow the production of new drugs based on DNA base sequences of genes or the structure of proteins coded for by these genes Ancestry (for understanding human origins & evolution) J WERBA – IB BIOLOGY 26

GENETIC CODE IS UNIVERSAL Command term = STATE 4.4.7 GENETIC CODE IS UNIVERSAL Command term = STATE Genes are able to be transferred between species because the genetic code is universal. This means that the amino acid sequence of proteins translated from them is unchanged. J WERBA – IB BIOLOGY 27

GENE TRANSFER Plasmid removed from bacterial cell 4.4.8 GENE TRANSFER Plasmid removed from bacterial cell plasmids are self-replicating rings of DNA Gene of interest removed from organism Gene amplified with PCR Gene of interest spliced into plasmid Gene and plasmid cut with same restriction enzyme This creates matching sticky ends like a puzzle Spliced (glued) together with DNA ligase Recombinant plasmid inserted into host cell The transgenic cells should express the new trait J WERBA – IB BIOLOGY 28

4.4.8 GENE TRANSFER J WERBA – IB BIOLOGY 29

4.4.8 GENE TRANSFER J WERBA – IB BIOLOGY 30

RECOMBINATION

GENETICALLY MODIFIED CROPS and ANIMALS 4.4.9 GENETICALLY MODIFIED CROPS and ANIMALS Crop plants and domestic animals have been modified over generations using selective breeding, to produce organisms with desirable characteristics. Recombinant DNA technology can now be used to alter the genetic make-up of organisms much more quickly. Some genetically modified organisms (GMOs) are already being produced. In 2006, 252 million acres of transgenic crops were grown globally. J WERBA – IB BIOLOGY 32

GENETICALLY MODIFIED CROPS and ANIMALS 4.4.9 GENETICALLY MODIFIED CROPS and ANIMALS J WERBA – IB BIOLOGY 33

GENETICALLY MODIFIED CROPS and ANIMALS Command term = STATE 4.4.9 GENETICALLY MODIFIED CROPS and ANIMALS Command term = STATE You need to know two examples of the current uses of genetically modified crops or animals Examples include: Bt corn produces a toxin (from Bacillus thuringiensis) that kills corn borers sheep that produce human blood clotting factor IX in their milk synthesis of beta-carotene in Golden RiceTM (vitamin A precursor so ups nutritional value) herbicide resistance in crops salt tolerance in plants delayed ripening in Flavr-Savr™ tomatoes bacteria use to produce insulin and clotting factor VIII J WERBA – IB BIOLOGY 34

EFFECTS OF GENETIC MODIFICATION 4.4.10 EFFECTS OF GENETIC MODIFICATION J WERBA – IB BIOLOGY 35

EFFECTS OF GENETIC MODIFICATION 4.4.10 EFFECTS OF GENETIC MODIFICATION Benefits: Higher crop yield (lessen food shortages) Higher yields = less land used for farming Reduction in use of pesticides Less expenditure, greater profits J WERBA – IB BIOLOGY 36

EFFECTS OF GENETIC MODIFICATION 4.4.10 EFFECTS OF GENETIC MODIFICATION Disadvantages: May have unknown health effects (allergy?) Toxin may kill non-pest species Cross-pollination to weeds Reduced biodiversity J WERBA – IB BIOLOGY 37

CLONE Command term = DEFINE 4.4.11 CLONE Command term = DEFINE Clone: a group of genetically identical organisms J WERBA – IB BIOLOGY 38

4.4.12 REPRODUCTIVE CLONING Scientists can use genetic technologies to clone whole organisms; this is known as reproductive cloning. Farm animals or crop plants with desirable features can be cloned, ensuring a constant food supply and high productivity. It is thought that reproductive cloning could also help to increase numbers of rare or endangered species. J WERBA – IB BIOLOGY 39

4.4.10 REPRODUCTIVE CLONING J WERBA – IB BIOLOGY 40

REPRODUCTIVE CLONING Cells are removed from two donor organisms: 4.4.12 REPRODUCTIVE CLONING Cells are removed from two donor organisms: Species 1 – cell remains intact Species 2 – nucleus is removed from cell Cells are fused using electricity (“electro-fusion”) Reconstructed cell is implanted into the womb of a Species 2 surrogate mother and forms an embryo A clone of the Species 1 organism will be born. J WERBA – IB BIOLOGY 41

4.4.12 REPRODUCTIVE CLONING J WERBA – IB BIOLOGY 42

4.4.13 THERAPEUTIC CLONING Scientists can use genetic technologies to produce organs, tissues and cells. This is known as therapeutic cloning or non- reproductive cloning. The process requires the use of stem cells and thus, is very controversial. J WERBA – IB BIOLOGY 43

ETHICS OF THERAPEUTIC CLONING 4.4.13 ETHICS OF THERAPEUTIC CLONING J WERBA – IB BIOLOGY 44

ETHICS OF THERAPEUTIC CLONING 4.4.13 ETHICS OF THERAPEUTIC CLONING Cloning happens naturally, for example monozygotic twins. Some may regard the in vitro production of two embryos from one to be acceptable. Others would see this as leading to the selection of those "fit to be cloned" and visions of "eugenics and a super-race". J WERBA – IB BIOLOGY 45

ETHICS OF THERAPEUTIC CLONING 4.4.13 ETHICS OF THERAPEUTIC CLONING Arguments for Therapeutic Cloning May be used to cure serious diseases (replacing bad cells with good ones) Stem cell research may pave the way for future discoveries Stem cells can be taken from aborted/unviable embryos or from the umbilical cord or adult source Cells are taken at a stage when the embryo has no nervous system and can arguably feel no pain Arguments against Therapeutic Cloning Involves the creation and destruction of human embryos Stem cells are capable of continued division and may develop into tumours More embryos are produced than are needed, so excess embryos are killed Alternative technologies may fulfil similar roles in the future (eg. reprogramming of differentiated cell lines) J WERBA – IB BIOLOGY 46

Sample questions Q1 State the name of the technique that is used to separate fragments of DNA according to their size, during DNA profiling. (1) The diagram below represents the results of a paternity investigation. Track A is the profile of the mother of a child, track B is the profile of the child and track C is the profile of a man who might be the father. Explain, using evidence from the diagram, whether this man is the father or not. (3) J WERBA – IB BIOLOGY 47

Sample questions A1 (gel) electrophoresis 1 (track C is) not (DNA from) the father; some bands on track B do not occur on A or C; these bands must be DNA inherited from the real father; band in track B that does not occur on A or C identified; Annotations to the gel may be used. 3 max J WERBA – IB BIOLOGY 48

Sample questions Q2 Outline the method that can be used to amplify small quantities of DNA to obtain large enough quantities for DNA profiling. (2) Q3 Describe a technique used for gene transfer. (5) Q4 Using a named example, discuss the benefits and harmful effects of genetic modification. (9) J WERBA – IB BIOLOGY 49

Sample questions A2 polymerase chain reaction/PCR; (DNA obtained from) blood/semen/hairs/other source of tissue; combined with necessary raw materials/one example of raw material; in thermal cycler / (PCR) machine; DNA replicated many times; J WERBA – IB BIOLOGY 50

Sample questions A3 restriction enzymes/endonucleases cut a small fragment of DNA from an organism; same restriction enzymes used to cut DNA of plasmid / e.g. E. coli; sticky ends are the same in both cases; fragment of DNA is inserted into the plasmid; spliced together by ligase; to make recombinant DNA/plasmids; recombinants can be inserted into host cell and cloned; J WERBA – IB BIOLOGY 51

Sample questions A4 genetic modification is when the DNA/genotype of an organism is artificially changed; genetic modification alters some characteristic/phenotype of the organism; named example with modification (e.g. salt tolerance in tomato plants); benefits: [5 max] allows crops to be grown where they would not grow naturally; provides more food; economic benefits; expands world’s productive farmland; reduces the need to clear rainforests to grow crops; lowers cost of production; less pesticides/fertilizers/chemicals needed so better for environment; harmful effects: [5 max] may be released into natural environment; may affect food chains / unintended effects on other organisms; may affect consumers e.g. allergies/health risks; unfair to smaller farmers who cannot compete; long-term effects are unknown; risk of cross-pollination; risk of long-term contamination of soil; J WERBA – IB BIOLOGY 52