Genetic Modification and Biotechnology PDF

Understandings: PCR can be used to amplify small amounts of DNA an artificial method of replicating DNA under lab conditions PCR occurs in a thermal cycler and uses variations in temperature to control the replication process: 1. Denaturation – DNA sample is heated to separ...

Understandings: PCR can be used to amplify small amounts of DNA an artificial method of replicating DNA under lab conditions PCR occurs in a thermal cycler and uses variations in temperature to control the replication process: 1. Denaturation – DNA sample is heated to separate it into two single strands (~95ºC for 1 min) 2. Annealing – DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min) 3. Elongation – A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min) Gel electrophoresis is used to separate proteins or fragments of DNA according to size a laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel ○ Smaller samples are less impeded by the gel matrix and hence will move faster through the gel ○ This causes samples of different sizes to separate as they travel at different speeds DNA profiling involves comparison of DNA a technique by which individuals can be identified and compared via their respective DNA profiles ○ Within the non-coding regions of an individual’s genome there exists satellite DNA – long stretches of DNA made up of repeating elements called short tandem repeats (STRs) ○ As individuals will likely have different numbers of repeats at a given satellite DNA locus, they will generate unique DNA profiles Genetic modification is carried out by gene transfer between species A gene determines a particular trait by encoding for a specific polypeptide in a given organism Because the genetic code is universal, an organism can potentially express a new trait if the appropriate gene is introduced into its genome The transfer of genes between species is called gene modification ○ the new organism created is called a transgenic Clones are groups of genetically identical organisms, derived from a single original parent cell Organisms that reproduce asexually will produce genetically identical clones ○ Additionally, mechanisms exist whereby sexually reproducing organisms can produce clones (e.g. identical twins) Cloning multicellular organisms requires the production of stem cells (differentiated cells cannot form other cell types) ○ Stem cells can be artificially generated from adult tissue using a process called somatic cell nuclear transfer (SCNT)  Many plant species and some animal species have natural methods of cloning Many species can reproduce asexually and hence possess natural methods of cloning ○ All bacteria, the majority of fungi and many species of protists reproduce asexually to produce genetic clones ○ While most plants reproduce sexually, they also possess methods of asexual reproduction (vegetative propagation) ○ Certain animal species can also reproduce asexually, via a variety of different mechanisms: Binary Fission The parent organism divides equally in two, so as to produce two genetically identical daughter organisms occurs in Planaria (flatworms) but is also common to bacteria and protists Budding Cells split off the parent organism, generating a smaller daughter organism which eventually separates from the parent occurs in Hydra but is also common to many species of yeast Fragmentation New organisms grow from a separated fragment of the parent organism common to starfish and certain species of annelid worms Parthenogenesis Embryos are formed from unfertilised ova (via the production of a diploid egg cells by the female) occurs in certain species of insect, fish, amphibians and reptiles Human Cloning Methods Identical twins (monozygotic) are created when a fertilised egg (zygote) splits into two identical cells, each forming an embryo Non-identical twins (dizygotic) are created when an unfertilised egg splits into two cells and each is fertilised by a different sperm Identical twins will be clones of one another (genetically identical), while non-identical twins will share 50% of the same DNA Animals can be cloned at the embryo stage by breaking up the embryo into more than one group of cells At a very early stage, embryonic cells can divide and become any type of tissue ○ These cells will differentiate to form all the different tissues comprising the organism If these embryonic cells are separated artificially in the laboratory, each group of cells will form cloned organisms ○ This separation of embryonic cells can also occur naturally to give rise to identical (monozygotic) twins The separated groups of cells are then implanted into the uterus of a surrogate to develop into genetically identical clones  This method of cloning is limited by the fact that the embryo used is still formed randomly via sexual reproduction and so the specific genetic features of the resulting clones have yet to be determined Methods have been developed for cloning adult animals using differentiated cells A more reliable method of artificial cloning involves somatic cell nuclear transfer ○ This involves replacing the haploid nucleus of an unfertilised egg with a diploid nucleus from an adult donor ○ The advantage of this technique is that it is known what traits the clones will develop This method of using differentiated cells to generate cloned embryos can be used for two main purposes: ○ Reproductive cloning: If the embryo is implanted into the uterus of a surrogate, a new cloned organism will develop ○ Therapeutic cloning: Embryonic cells can be induced to differentiate to create specific tissues or organs for transplantation Applications: Use of DNA profiling in paternity and forensic investigations DNA profiling is commonly used in criminal investigations and to settle paternity disputes ○ A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR ○ Satellite DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments ○ Fragment length will differ between individuals due to the variable length of their short tandem repeats ○ The fragments are separated using gel electrophoresis and the resulting profiles are compared Paternity Testing: ○ Children inherit half their chromosomes from each parent and thus should possess a combination of parental fragments Gene transfer to bacteria using plasmids makes use of restriction endonucleases and DNA ligase 1. Isolation of gene and vector (by PCR) 2. Digestion of gene and vector (by restriction endonuclease) 3. Ligation of gene and vector (by DNA ligase) 4. Selection and expression of transgenic construct Assessment of the potential risks and benefits associated with genetic modification of crops Production of cloned embryos produced by somatic cell nuclear transfer Skills: Design of an experiment to assess one factor affecting the rooting of stem cuttings There are a variety of factors that will influence successful rooting of a stem cutting, including: ○ Cutting position (whether cutting occurs above or below a node, as well as the relative proximity of the cut to the node) ○ Length of cutting (including how many nodes remain on the cutting) ○ Growth medium (whether left in soil, water, potting mix, compost or open air) ○ The use and concentration of growth hormones ○ Temperature conditions ○ Availability of water ( ○ Other environmental conditions (including pH of the soil and light exposure) Analysis of examples of DNA profiles Analysis of data on risks to monarch butterflies of Bt crops Bt corn is a genetically modified maize that incorporates an insecticide producing gene from the bacterium Bacillus thuringiensis  ○ This insecticide is lethal to certain types of larvae, particularly the European corn borer which would otherwise eat the crop Concerns have been raised that the spread of Bt corn may also be impacting the survival rates of monarch butterflies ○ While monarch butterfly larva feed exclusively on milkweed, wind-borne pollen from Bt corn may dust nearby milkweeds ○ Caterpillars exposed to Bt pollen were found to have eaten less, grew more slowly and exhibited higher mortality rates