3.5 Genetic modification and biotechnology Flashcards
What is PCR and what is it used for?
- The polymerase chain reaction (PCR) is an artificial method of replicating DNA under laboratory conditions
- The PCR technique is used to amplify large quantities of a specific sequence of DNA from an initial minute sample
- Each reaction cycle doubles the quantity of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (230)
Describe the stages of PCR
PCR occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:
- Denaturation – DNA sample is heated to separate it into two single strands (~95ºC for 1 min)
- Annealing – DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min)
- Elongation – A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min)
What happens once large quantities of DNA have been created through PCR?
Other laboratory techniques are used to isolate and manipulate the sequences.
Diagram showing an overview of the PCR cycle
What is gel electrophoresis?
A laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size
Give an overview of how gel electrophoresis works
- 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
Diagram of the gel electrophoresis apparatus
Are DNA and protein separations the same?
While both DNA and proteins are separated according to the same basic process, differences exist between the two protocols
Explain how DNA separation using gel electrophoresis works
- DNA may be cut into fragments using restriction endonuclease – different DNA samples will generate different fragment lengths
- Fragments separate because DNA is negatively charged due to the presence of a phosphate group (PO43–) on each nucleotide
- DNA samples are placed into an agarose gel and fragment size is calculated by comparing them against known industry standards
- Specific sequences can be identified by incorporating a complementary radiolabelled hybridization probe, transferring the separated sequences to a membrane, and then visualizing via autoradiography (Southern blotting)
Diagram of agarose gel electrophoresis (DNA)
Explain how protein separation using gel electrophoresis works
-Proteins may be folded into a variety of shapes (affecting size) and have positive and negative regions (no clear charge)
-Proteins must first be treated with an anionic detergent (SDS) in order to linearise and impart a uniform negative charge
-Protein samples are placed into a polyacrylamide gel and sizes are compared against known industry standards
-Separated proteins are transferred to a membrane and then target proteins are identified by staining with specific monoclonal antibodies (Western blotting)
Diagram of polyacrylamide gel electrophoresis (proteins)
What is DNA profiling and how does it work?
-DNA profiling is 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
Diagram of comparative STR lengths at two specific loci
What are the uses of DNA profiling?
For criminal investigations (forensics) and to settle paternity disputes (among other uses)
Describe the procedure of DNA profiling
-A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR
-Satellite DNA (with STR sequences) is 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
How can DNA profiling be used in forensic investigations?
-Suspects should be a complete match with the DNA sample taken from the crime scene if a conviction is to occur
-The number of loci used to generate a unique profile depends on the size of the population being compared
-E.g. America (population: ~ 320 million) uses 13 loci for comparison; Australia (population: ~ 25 million) uses only 9 loci
How can DNA profiling be used in paternity testing?
-Children inherit half their chromosomes from each parent and thus should possess a combination of parental fragments
-In other words, all fragments produced in the child should also be produced by either the mother or father
Diagram of the DNA profiles of DNA found at a crime scene
In this DNA profile, who is the biological father?
Father 2
Key points about analyzing DNA evidence
-To find a match between DNA evidence and an individual (suspect or victim), every band in the gel must be an identical match.
-To determine paternity, start by matching the DNA fragments from the child with the mother’s profile. For the child, every band that does not match the mother must have a match in the father’s profile.
What conclusion can be made from the following DNA profiling evidence?
-Both children are related to both parents.
-Once you have identified all fragment lengths in the children’s profiles that can be matched to the mother’s profile, the remaining fragment lengths can all be matched to the father’s profile. This is true for both children.
What does a gene determine and how?
It determines a particular trait by encoding a specific polypeptide in a given organism.
Why is gene transfer possible?
Because the genetic code is (almost) universal, an organism can potentially express a new trait if the appropriate gene is introduced into its genome.
What is genetic modification and what is a transgenic organism?
The transfer of genes between species is called gene modification, and the new organism created is called a transgenic.
Diagram showing bacteria producing human insulin (Bioninja)
Diagram showing a genetic modification of a bacterium to produce human insulin
What are the steps of gene transfer?
- Isolation of gene and vector (by PCR)
- Digestion of gene and vector (by restriction endonuclease)
- Ligation of gene and vector (by DNA ligase)
- Selection and expression of the transgenic construct
Explain step 1 of gene transfer: Isolating gene and vector (by PCR)
- DNA can be isolated from cells by centrifugation – whereby heavier components such as nuclei are separated
- The gene of interest can then be specifically amplified via the polymerase chain reaction (PCR)
- Gene sequences can also be generated from mRNA using reverse transcriptase – these DNA sequences (cDNA) lack introns
- A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell
- Bacterial plasmids are commonly used as vectors because they are capable of autonomous self-replication and expression
- These plasmids may be modified for further functionality (e.g. selection markers, reporter genes, inducible expression promoters)
- Other types of vectors include modified viruses and artificial chromosomes
Diagram showing the common features of a typical plasmid vector
Explain step 2 of gene transfer: digestion with restriction enzymes
- In order to incorporate a gene of interest into a vector, both must be cut with restriction enzymes at specific recognition sites
- Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs)
- Scientists will often cleave the vector and gene with two different ‘sticky end’ restriction endonucleases (double digestion) to ensure the gene is inserted in the correct orientation and to prevent the vector from re-annealing without the desired insert
Diagram showing ‘sticky end’ vs. ‘blunt end’ restriction enzymes
Explain step 3 of gene transfer: Ligation of gene and vector (by DNA ligase)
-The gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases
-This occurs because the sticky ends of the gene and vector overlap via complementary base pairing
-The gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct
-DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond
Diagram of the formation of a recombinant construct
Explain step 4 of gene transfer: selection and expression of the transgenic construct
-The recombinant construct (including the gene of interest) is finally introduced into an appropriate host cell or organism
-This process can be achieved in a variety of ways and is called transfection (for eukaryotes) or transformation (for prokaryotes)
-Antibiotic selection is commonly used in order to identify which cells have successfully incorporated the recombinant construct
-The plasmid vector contains an antibiotic resistance gene, so only transgenic cells will grow in the presence of antibiotic
-Transgenic cells, once isolated and purified, will hopefully begin expressing the desired trait encoded by the gene of interest
Diagram showing the isolation of transgenic cells via antibiotic selection
Why are plasmids useful in genetic modification of bacteria?
They are easily transferred between bacterial cells and are small enough to manipulate easily.
What is used to seal a target gene into a bacterial plasmid?
DNA ligase
What separates genetic modification from earlier methods of manipulating offspring such as selective breeding?
Genetic modification transfers genes between different species.
Describe the results of large-scale studies about GM crops
-They have shown mixed impacts from GM crops.
-In some cases, GM crops have reduced the need for damaging pesticides; in other cases, however, they have actually increased their use.
-Many nations have established committees that evaluate the potential risks of experiments involving the genetic modification of organisms.
-Some countries ban the cultivation of GM crops but allow them to be imported, while others grow many GM crops.
Benefits of GMO crops
-Introduction of new positive traits to the crop
-Economic advantages
-Environmental advantages
Risks of GMO crops
-Ecosystem damage
-Increasing monoculture
-Corporate control over food supply
-Human health concerns
Explain how the introduction of new positive traits to the crop is a benefit of GMOs and give an example
-Almost any trait desired can be developed: increased vitamin content, drought or disease resistance, cold or salt tolerance, allergen reduction
-Golden corn contains three added genes to provide precursors of vitamin A and could prevent hundreds of thousands of cases of childhood blindness per year caused by vitamin A deficiency.
Explain how economic advantages are a benefit of GMOs and give an example
-GMO crops can provide a greater yield in a variety of ways: longer shelf-life, less loss of food to environmental issues like disease, herbivores, and frost; greater profit, and lower prices for consumers.
-The use of Bt corn (added gene to resist pests) can produce 20%–40% more corn per unit of land than conventional corn.
Explain how environmental advantages are a benefit of GMOs
-Higher yields mean less land is needed for farming, leaving a more natural ecosystem. Less pesticide needs to be sprayed.
-There is a documented drop in sprayed pesticides on farms using Bt potatoes. (However, if the pests evolve resistance this advantage may disappear.)
Explain how ecosystem damage is a risk of GMOs and give an example
-GMO species may accidentally outcompete native species, kill or damage non-pest species, or have other harmful effects. Cross-pollination could introduce new genes to weed species.
-GM soybeans resistant to herbicide led to a documented increase in the amount of sprayed herbicide used.
Explain how increasing monoculture is a risk of GMOs and give an example
-GMO crops have very low biodiversity, having been cloned from an originally modified plant. There is little resistance if a new threat emerges.
-Corn earworm and rootworm pests have developed resistance to Bt in corn. (Low biodiversity is also common in conventional crops but is not necessarily so.)
Explain how corporate control over food supply is a risk of GMOs and give an example
-Not all nations and farmers can access this technology. Increased pest attacks on traditional farms, and increased inequality between large farms and family or subsistence farms.
-Monsanto, the major GMO company, sues farmers for planting GM seeds saved from the previous season, which Monsanto forbids.
-Some subsistence farmers near large GMO farms have very high numbers of pests in their fields (the refuge phenomenon), causing extensive damage.
Explain how human health concerns are a risk of GMOs and give an example
-Exposure to new genes and their protein products could cause unknown impacts and damage, including possible allergic reactions and damage to mutualistic probiotic bacteria in the digestive system.
-An attempt to improve the nutritional value of soybeans by adding a Brazil nut protein showed that people allergic to nuts were also allergic to GM soybeans.
-Many studies have found that various GM crops do not damage organs and are digested without issue, but the field is rapidly changing and continued research is necessary.
It is important to remember that ___ being transferred during genetic modification matters greatly when considering possible impacts.
The nature of the genes
When a farmer replaces a field of potatoes of a single cultivar (all the same type) with a genetically modified cultivar where potatoes produce a type of insecticide called Bt toxin, which is the most likely result?
-An increase in resistance to Bt toxin in the insect populations over time.
-This situation will increase natural selective pressures on the insects. Those with genetic variations that allow them to withstand higher doses of Bt toxin will survive longer and have offspring that share their resistance. Over time, the insecticide produced by the potatoes may fail to control the insects.
What is Bt corn and how can it be harmful?
-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
What concerns have been raised about the effect of Bt corn?
-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
Diagram showing the relationship between Bt corn and the monarch butterfly
Describe the data that investigated the effect of Bt corn on monarch butterflies
-In 1999, a preliminary study was conducted investigating the association between exposure to Bt corn pollen and survivor rates among monarch caterpillars:
-Monarch caterpillars were fed milkweed leaves that had been dusted with pollen from Bt corn (to simulate spread via wind)
-Growth and mortality rates were compared against caterpillars fed on non-dusted leaves or leaves dusted with non-GM pollen
-Caterpillars exposed to Bt pollen were found to have eaten less, grew more slowly, and exhibited higher mortality rates
Graph showing the effect of Bt pollen on survival rates of monarch butterflies (laboratory conditions)
Why have some scientists questioned the 1999 preliminary study about the effects of Bt corn?
-Some scientists suggested that these results may lack validity as they do not accurately reflect natural conditions:
-There were higher amounts of Bt pollen on the leaves than would be found naturally (e.g. rain would diminish build-up)
-Larvae were restricted in their diet (in the field, larvae could feasibly avoid eating pollen-dusted leaves)
Describe the second study on the effect of Bt corn
-A second study was conducted comparing the survivor rates of monarch butterflies based on proximity to Bt corn fields:
-There was no significant increase in mortality when monarch larvae were placed in or near an actual Bt cornfield
-From this, it was concluded that exposure to Bt pollen poses no significant risk to monarch butterfly populations
Diagram showing the effect of Bt pollen on survival rates in monarch butterflies (field conditions)
What is a stem cutting?
A separated portion of a plant stem that can regrow into a new independent clone via vegetative propagation
What are the features of stems and how are stem cuttings used?
-All stems possess nodes, from which a leaf, branch, or aerial root may grow – the region between nodes are called internodes
-Stem cuttings are typically placed in the soil with the lower nodes covered and the upper nodes exposed
-Stem cutting is a common method employed to rapidly propagate plant species (including sugar cane, grapes and roses)
What factors will influence the success of rooting of a stem cutting?
-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 the soil, water, potting mix, compost, or open-air)
-The use and concentration of growth hormones (e.g. IAA, IBA, and NAA promote the formation of adventitious roots)
-Temperature conditions (most cuttings grow optimally at temperatures common to spring and summer)
-Availability of water (either in the form of groundwater or humidity)
-Other environmental conditions (including pH of the soil and light exposure)
Diagram of germination of stem cuttings
What are clones and how do they come about?
-Clones are groups of genetically identical organisms or a group of cells 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)
What does cloning of multicellular organisms require?
-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)
Explain how somatic cell nuclear transfer can produce cloned embryos
-Somatic cell nuclear transfer is a method by which cloned embryos can be produced using differentiated adult cells
-Somatic cells are removed from the adult donor and cultured (these cells are diploid and contain the entire genome)
-An unfertilized egg is removed from a female adult and its haploid nucleus is removed to produce an enucleated egg cell
-The enucleated egg cell is fused with the nucleus from the adult donor to make a diploid egg cell (with the donor’s DNA)
-An electric current is then delivered to stimulate the egg to divide and develop into an embryo
-The embryo is then implanted into the uterus of a surrogate and will develop into a genetic clone of the adult donor
Diagram showing somatic cell nuclear transfer
Give an overview of 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
Give examples of animal cloning methods
-Binary fission
-Budding
-Fragmentation
-Parthenogenesis
Explain binary fission
-The parent organism divides equally in two, so as to produce two genetically identical daughter organisms
-This method of cloning occurs in Planaria (flatworms) but is also common to bacteria and protists (e.g. euglena, amoeba)
Explain budding
-Cells split off the parent organism, generating a smaller daughter organism which eventually separates from the parent
-This method of cloning occurs in Hydra but is also common to many species of yeast
Explain fragmentation
-New organisms grow from a separated fragment of the parent organism
-This method of cloning is common to starfish and certain species of annelid worms
Explain parthenogenesis
-Embryos are formed from unfertilized ova (via the production of a diploid egg cell by the female)
-This method of cloning occurs in certain species of insects, fish, amphibians, and reptiles
Diagram showing the methods of animal cloning
Explain how plants can clone themselves
-Plants have the capacity for vegetative propagation, whereby small pieces can be induced to grow independently
-This is because adult plants possess meristematic tissue capable of cellular differentiation (totipotent)
Give examples of vegetative propagation
-Virtually all types of roots and shoots are capable of vegetative propagation
-Garlic and onion bulbs are modified plant leaves – all the bulbs in a group are genetically identical
-Underground stems (e.g. potato tubers) can form new plants which are genetically identical to the parent plant
-Certain plants can form horizontal stems called runners (or stolons) that grow roots and develop into clones
Describe asexual reproduction in plants
-Some plants (mainly algae, mosses, and ferns) can reproduce asexually by producing spores
-Spores are also produced by certain types of bacteria and fungi
Diagram of vegetative propagation via plant stolons
Explain natural cloning in humans
-Even human beings are capable of creating genetic clones through natural means
-Identical twins (monozygotic) are created when a fertilized egg (zygote) splits into two identical cells, each forming an embryo
-Non-identical twins (dizygotic) are created when an unfertilized egg splits into two cells and each is fertilized 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
Diagram of monozygotic vs. dizygotic twins
Explain how animals can be cloned at the embryo stage
-At a very early stage, embryonic cells retain pluripotency (meaning they 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 separation of embryonic cells has to happen early in the developmental cycle, ideally around the 8-cell stage (morula)
-The separated groups of cells are then implanted into the uterus of a surrogate to develop into genetically identical clones
What is a limitation of the separation of embryonic cells artificially
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.
Diagram of cloning via embryonic division
Explain somatic cell nuclear transfer
-A second and more reliable method of artificial cloning involves somatic cell nuclear transfer (SCNT)
-This involves replacing the haploid nucleus of an unfertilized 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 (they are genetically identical to the donor)
What is somatic cell nuclear transfer (SCNT) used for?
-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
Diagram comparing reproductive and therapeutic cloning
What happens to the unfertilized egg used in somatic-cell nuclear transfer?
Its nucleus is replaced by the nucleus of the differentiated body cell.
