REVISION: GENETIC MODIFICATION and BIOTECHNOLOGY Biologists have developed techniques for artificial manipulation of DNA, cells and organisms. Topic 3.5 IB Biology Miss Werba

RESTRICTION ENZYMES Also called restriction endonucleases Cut a DNA molecule at a particular place The enzyme "scans" a DNA molecule, looking for a particular sequence. Create “sticky ends” with known sequences that can be utilised for genetic engineering eg. HindIII, EcoR1 J WERBA – IB BIOLOGY 2

REVERSE TRANSCRIPTASE Reverses transcription! Enzymes which can make DNA from RNA DNA formed is called copy DNA (cDNA) cDNA can be made double-stranded so it can be transferred to another organism J WERBA – IB BIOLOGY 3

DNA POLYMERASE Used to make DNA / multiple copies of DNA Allows single-stranded cDNA to be made double- stranded Allows small DNA samples to be amplified for testing eg. DNA from an embryo during aminocentesis, DNA from a crime scene J WERBA – IB BIOLOGY 4

DNA LIGASE Enzyme used for joining pieces of DNA together ie. genetic “glue” Allows an isolated / created piece of DNA to be joined to the DNA of a recipient organism (plasmid). J WERBA – IB BIOLOGY 5

GENE PROBES Single-stranded DNA used to identify genes Made using reverse transcriptase Are radioactively labelled so that they can be detected when they bind to a chromosome / segment J WERBA – IB BIOLOGY 6

MARKER GENES Genes close to the gene being studied Chosen because they produce an obvious phenotype that can be easily identified Presence of the marker indicates that the neighbouring gene has probably been inherited. Gene transfer: a plasmid would be introduced into the recipient organism with the desired gene and its marker. J WERBA – IB BIOLOGY 7

POLYMERASE CHAIN REACTION (PCR) A way to amplify (produce large quantities) of a specific target sequence of DNA Useful when only a small amount of DNA is available eg. crime scene samples Process of PCR: Denaturation: heat the DNA  separates strands Annealing: attach DNA primers  highlights target sequence Elongation: use DNA polymerase  replicates the sequence One cycle  yields 2 DNA copies J WERBA – IB BIOLOGY 8

POLYMERASE CHAIN REACTION (PCR) J WERBA – IB BIOLOGY 9

GEL ELECTROPHORESIS Used to separate proteins / DNA fragments according to size Process: DNA cut 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 10

GEL ELECTROPHORESIS J WERBA – IB BIOLOGY 11

THINGS TO COVER U.3 U.4 U.5 U.6 U.7 U.8 Statement Guidance DNA profiling involves comparison of DNA. U.4 Genetic modification is carried out by gene transfer between species. U.5 Clones are groups of genetically identical organisms, derived from a single original parent cell. U.6 Many plant species and some animal species have natural methods of cloning. U.7 Animals can be cloned at the embryo stage by breaking up the embryo into more than one group of cells. U.8 Methods have been developed for cloning adult animals using differentiated cells. J WERBA – IB BIOLOGY 12

THINGS TO COVER A.1 A.2 A.3 A.4 S.1 S.2 S.3 Statement Guidance NOS 4.8 Use of DNA profiling in paternity and forensic investigations. Students should be able to deduce whether or not a man could be the father of a child from the pattern of bands on a DNA profile. A.2 Gene transfer to bacteria using plasmids makes use of restriction endonucleases and DNA ligase. A.3 Assessment of the potential risks and benefits associated with genetic modification of crops. A.4 Production of cloned embryos produced by somatic-cell nuclear transfer. Dolly can be used as an example of somatic-cell transfer S.1 Design of an experiment to assess one factor affecting the rooting of stem-cuttings. A plant species should be chosen for rooting experiments that forms roots readily in water or a solid medium. S.2 Analysis of examples of DNA profiles. S.3 Analysis of data on risks to monarch butterflies of Bt crops. NOS 4.8 Assessing risks associated with scientific research. J WERBA – IB BIOLOGY 13

DNA PROFILING Also called DNA fingerprinting DNA profiling allows individuals to be identified on the basis of their DNA profiles. Can target short tandem repeats (STRs) in the non- coding regions of an individual's genome Repeating sequences removed and cut with a variety of restriction enzymes. Unique fragment profiles will be generated and can be compared using gel electrophoresis. eg. used to determine paternity, used in forensic investigations J WERBA – IB BIOLOGY 14

DNA PROFILING Paternity testing Forensic investigation J WERBA – IB BIOLOGY 15

GENE TRANSFER Genetic modification is carried out by gene transfer. Possible because the genetic code is universal. Process: Plasmid removed from bacterial cell Gene of interest removed with restriction enzyme Gene amplified with PCR Plasmid cut with same restriction enzyme to create sticky ends Gene spliced (glued) into plasmid with DNA ligase Recombinant plasmid inserted into host cell The transgenic cells should express the new trait J WERBA – IB BIOLOGY 16

GENE TRANSFER J WERBA – IB BIOLOGY 17

GENETICALLY MODIFIED CROPS and ANIMALS Gene transfer (Recombinant DNA technology) can be used to alter the genetic make-up of organisms Produces genetically modified organisms (GMOs) eg. 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 herbicide resistance in crops salt tolerance in plants bacteria use to produce insulin and clotting factor VIII J WERBA – IB BIOLOGY 18

Bt CORN Potential Benefits Potential Harmful Effects New characteristics can be introduced much faster than through selective breeding Can ↑ productivity of food production (requires less land for same yield) ↓ use of pesticides, ↓ economic cost of farming Allows farming in regions that may not have been viable, ↓ need for deforestation Potential Harmful Effects May have unknown harmful effects (eg. allergy to toxin) May compete with native plant species Possibility of cross pollination with weeds and be hard to control ↓ genetic variation / biodiversity (corn borer may play a crucial role in local ecosystem) J WERBA – IB BIOLOGY 19

Bt CORN & MONARCH BUTTERFLIES A gene from a bacterium (Bacillus thuringiensis) has been transferred to maize to protect them from corn borers. The gene codes for a bacterial protein called Bt toxin that kills corn borers feeding on the maize. Insects that are not pests could be killed accidentally. J WERBA – IB BIOLOGY 20

Bt CORN & MONARCH BUTTERFLIES eg. Maize pollen containing the toxin may blow onto wild plants (eg. milkweed) growing near the maize. Insects feeding on the milkweed, including Monarch butterfly caterpillars, will therefore be affected, even if they do not feed on the maize. Milkweed is the only plant used by Monarch butterflies to lay their eggs, and Monarch caterpillars to eat. As of January 2014, the Monarch population is at its lowest point in history. J WERBA – IB BIOLOGY 21

CLONING Clone: a group of genetically identical organisms, derived from a single original parent cell. Examples: Natural cloning monozygotic twins use of plant cuttings  you need to be able to design an experiment to assess one factor affecting the rooting of stem- cuttings asexual reproduction Reproductive cloning Therapeutic cloning J WERBA – IB BIOLOGY 22

REPRODUCTIVE CLONING Cloning of whole organisms Can be cloned at the embryo stage by breaking up the embryo into more than one group of cells Somatic cell nuclear transfer (SCNT) was used to clone Dolly the Sheep J WERBA – IB BIOLOGY 23

REPRODUCTIVE CLONING Process: 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 24

REPRODUCTIVE CLONING J WERBA – IB BIOLOGY 25

THERAPEUTIC CLONING Production of organs, tissues and cells Also known as non-reproductive cloning Process requires the use of stem cells and thus, is very controversial. J WERBA – IB BIOLOGY 26