3.5 Genetic modification and biotechnology Applications: Use of DNA profiling in paternity and forensic investigations Gene transfer to bacteria with plasmids using restriction endonucleases and DNA ligase Assessment of the potential risks and benefits associated with genetic modification of crops Production of cloned embryos by someatic cell nuclear transfer Understanding: Gel electrophoresis is used to separate proteins of fragments of DNA according to size PCR can be used to amplify small amounts of DNA DNA profiling involves comparison of DNA Genetic modification is carried out by gene transfer between species Clones are groups of genetically identical organisms, derived from a single original parent cell Many plant species and some animal species have natural methods of cloning Animals can be cloned at the embryo stage by breaking up the embryo into more than one group of cells Methods have been developed for cloning adult animals using differentiated cells Skills: Design of an experiment to assess one factor affecting the rooting of stem cuttings Analysis of examples of DNA profiles Analysis of data on risks to monarch butterflies of Bt crops Nature of science: Assessing risks associated with scientific research: scientists attempt to assess the risks associated with genetically modified crops or livestock

Polymerase Chain Reaction Amplify small amounts of DNA Just need a single molecule of DNA to make millions of copies Able to study DNA without using up the whole sample DNA from fossils DNA from crime scene (hair, semen or blood)

PCR Answer these questions What are the ingredients for PCR? What are primers? Why must the mixture be heated/cooled to about 95°C? 55-60°C? 72°C?

Answers 1. Template DNA, DNA primers, Deoxynucleotide triphosphates, thermophilic polymerase with a buffer 2. Primers start the chain reaction 3. 95 = DNA strands separate as hydrogen bonds break 55-60 = Primers bind to single DNA strands 75 = optimum temperature for DNA polyermase enzyme

Gel electrophoresis Separate proteins according to size In an electric field Samples placed into wells in a gel Electrical field applied Charged molecules move through the gel Small fragments move faster than large ones

Gel Electrophoresis Activity Go to: http://learn.genetics.utah.edu/content/labs/gel/ Answer the following questions: What is the gel made up of? What is the purpose of the buffering solution that the gel is immersed in? Why are known DNA fragment lengths/size standards needed? How does the gel separate DNA strands of different lengths?

Powdered agarose, buffer (Jell-o) Answers Powdered agarose, buffer (Jell-o) Salt water solution that allows electrical charges to move through the gel Compare your bands to bands of known length to help you identify the length of DNA DNA moves through gel repelled by negative charge – smaller moves further away

DNA profiling Sample of DNA obtained Sequences selected and copied by PCR Copied DNA split into fragments using enzymes Fragments separated using gel electrophoresis Produces a pattern of bands that is always the same with DNA from an individual Compare profiles of individuals to see which bands are similar or different

Compare DNA at a crime scene with suspects DNA

Paternity tests Men claim they are not the father Mother has multiple partners and isn’t sure Child wishes to prove they are an heir

Genetic modification Transfer of genes between species Genetic code is universal = amino acid sequences translated from genetic code is unchanged Same polypeptide is produced

Genetic modification Gene for making human insulin to bacteria Goats produce milk that contains spider silk Many GM crops

Salmon that grows twice as fast as normal

Contain vaccines for diseases Vaccine bananas Contain vaccines for diseases

Low methane cows 25% less methane – less impact on the environment

Involves the use of plasmids, DNA ligase and restriction endonucleases Gene transfer Involves the use of plasmids, DNA ligase and restriction endonucleases

Restriction endonuclease enzyme cuts DNA DNA ligase fixes the strands together

Multiply bacteria containing gene

Separate and purify human insulin Human insulin can then be used by diabetic patients

Enzyme also cuts bacterial DNA This produces a lot of insulin for human use Separate and purify insulin Insert insulin gene using DNA ligase Cut out insulin using restriction endonuclease Bacteria now contains insulin gene Locate the cell that contains insulin Multiply bacteria in a fermenter

3. Enzyme also cuts bacterial DNA 7 3. Enzyme also cuts bacterial DNA 7. This produces a lot of insulin for human use 8. Separate and purify insulin 4. Insert insulin gene using DNA ligase 2. Cut out insulin using restriction endonuclease 5. Bacteria now contains insulin gene 1. Locate the cell that contains insulin 6. Multiply bacteria in a fermenter

Advantages and disadvantages (summarise page 192-194 all boxes!) What is it How is it done What is it useful for (Use examples) Advantages and disadvantages (summarise page 192-194 all boxes!)

Benefits Risks Environmental Health Agricultural Fewer chemicals used and leaked into the environment Less eutrophication Out crossing of genes Alter ecosystems Affect food chains If some plants are pesticide resistant – a lot of pesticides can be used then just to be sure pests are dead Harm to other organisms Health Increase mineral/vitamin content Decrease starvation (Sustainable way to feed the world) Allergic reactions (do not know where a gene comes from) Fewer nutrients Ethical issues Agricultural Reduce pesticides (spend less money) Increased yields More aesthetically pleasing Affect other organisms other than the target pests

Bt corn Produces a toxin to protect them from corn pests Wind pollinated plant Pollen which also contains toxin blown away Monarch butterflies die

For or against Many studies carried out Studies need to be carried out over large periods of time to see if there is a significant issue Many studies accused of being biased for/against GMOs GM good = GM companies GM bad = organic farmers

What is a clone? A clone is genetically identical to its “parent” (single original parent)

Two types of reproduction SEXUAL Genetically different offspring ASEXUAL Genetically identical offspring

Sexual Reproduction HUMANS GAMETES Sperm cell: Egg cell: Each gamete contains 23 single chromosomes Half the number of that in a normal cell (23 pairs) These are haploid cells

Sexual Reproduction FERTILISATION Gametes fuse together Form a cell with 23 pairs of chromosomes This is a Diploid cell Offspring inherits features from both parents Variation in offspring

Advantages SEXUAL REPRODUCTION Variation in offspring means they are able to Adapt to surroundings Evolve

Asexual Reproduction An ordinary cell can make a new cell by dividing in two New cell has exactly the same information as parent cell No fertilisation between male and female gametes Some plants and bacteria produce offspring asexually (some animals do too when no males around)

Advantages ASEXUAL REPRODUCTION Very quick Bacteria can produce offspring in 20 mins If already adapted to habitat – good to be identical

Cloning animal embryos Break up embryo into more than one group of cells All cells in an early animal embryo are able to develop into any type of tissue

Cloning animal embryos Sometimes this happens naturally Can also be done artificially Cannot tell whether the individual has the desired characteristics

Cloning Adults Harder than cloning embryos as cells have already differentiated into different tissue cells

Dolly the sheep Somatic-cell nuclear transfer 1. Adult cells taken from udder of Finn Dorset ewe and grown in the lab (udder contains some stem cells)

Dolly the sheep 2. Cells grown in a medium of nutrients Genes in the cells become inactive Pattern of differentiation was lost

Dolly the sheep 3. Unfertilised eggs taken from ovaries of a Scottish Blackface ewe Nuclei were removed from these eggs

Dolly the sheep 4. Electric pulse used to fuse the two cells together 10% of the fused cells develop into an embryo

Dolly the sheep 5. Emrbyos injected when around 7 days old into the uteri of surrogate mother ewes One out of 29 was successful = DOLLY! Who does Dolly look like?

Dolly the sheep As it is the nucleus from a body cell of the Finn Dorset ewe Dolly is a clone of her.