3.5 genetic modification Flashcards
1
Q
what is pcr?
A
- polymerase chain reaction
- artificial method of replicating dna under laboratory conditions
- used to amplify large quantities of a specific sequence of dna from an initial minute sample
- each reaction cycle doubles the amount of dna – a standard pcr sequence of 30 cycles creates over 1 billion copies (2^30)
STAGES OF PCR
- occurs in a thermal cycler and uses variations in temperature to control replication process via 3 steps:
- denaturation – dna sample is heated to separate it into 2 single strands (~95ºC for 1 min) [strands separate]
- annealing – dna primers attach to 3’ ends of the target sequence (~55ºC for 1 min) [primers bind template]
- elongation / extension – heat-tolerant dna polymerase (Taq) binds to primer and copies strand (~72ºC for 2 min) [synthesize new strand]
- once large quantities of dna have been created, other laboratory techniques are used to isolate and manipulate the sequences
- pcr components: dna sample, primers (artificial, thus no need for primase), nucleotides, Taq polymerase, mix buffer, pcr tube
2
Q
what is gel electrophoresis?
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
- negatively charged dna moves towards positive electrode
- smaller samples are less impeded by gel matrix and hence will move faster through gel
- causes samples of different sizes to separate as they travel at different speeds
- while both dna and proteins are separated according to the same basic process, differences exist between the 2 protocols
3
Q
what are the differences in the gel electrophoresis process for proteins and dna?
A
OVERALL PROCEDURE:
- 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 gel matrix and hence will move faster through gel
- causes samples of different sizes to separate as they travel at different speeds
DNA SEPARATION
- 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 presence of a phosphate group (PO4 3–) on each nucleotide
- dna samples are placed into an agarose gel and fragment size calculated by comparing against known industry standards
- specific sequences can be identified by incorporating a complementary radiolabelled hybridisation probe, transferring the separated sequences to a membrane and then visualising via autoradiography (southern blotting)
PROTEIN SEPARATION
- 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 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)
4
Q
how does dna profiling work?
A
- technique by which individuals can be identified and compared via their respective dna profiles
- within 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)
- individuals will likely have different numbers of repeats at a given satellite dna locus, thus they will generate unique dna profiles
5
Q
when is dna profiling used?
A
- commonly used in criminal investigations (forensics) and to settle paternity disputes
common procedure:
- dna sample collected (e.g. from blood, semen, saliva, etc.) and then amplified using pcr
- satellite DNA (with STR [short tandem repeats] sequences) are cut with specific restriction enzymes to generate fragments
- fragment length will differ between individuals due to variable length of their short tandem repeats
- fragments are separated using gel electrophoresis and the resulting profiles are compared
FORENSIC INVESTIGATIONS
- suspects should be a complete match with dna sample taken from crime scene if a conviction is to occur
- number of loci used to generate a unique profile depends on the size of population being compared
PATERNITY TESTING
- children inherit half their chromosomes from each parent and thus should possess a combination of parental fragments
- all fragments produced in child should also be produced by either mother or father
6
Q
what is gene modification?
A
- gene determines a particular trait by encoding for a specific polypeptide in a given organism
- because genetic code is (almost) universal, an organism can potentially express a new trait if appropriate gene is introduced into its genome
- transfer of genes between species is called gene modification, and new organism created is called a transgenic
7
Q
outline the process of gene transfer
A
- summarised in 4 key steps:
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
- ISOLATION OF GENE AND VECTOR (BY PCR)
- dna can be isolated from cells by centrifugation – whereby heavier components such as nuclei are separated
- 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
- 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 - DIGESTION WITH RESTRICTION ENZYMES
- 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 2 different ‘sticky end’ restriction endonucleases (double digestion) to ensure gene is inserted in correct orientation and to prevent vector from re-annealing without desired insert - LIGATION OF VECTOR AND INSERT
- gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases
- occurs because sticky ends of gene and vector overlap via complementary base pairing
- gene and vector are then spliced together by the enzyme dna ligase to form a recombinant construct
- dna ligase joins vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond - SELECTION AND EXPRESSION
- recombinant construct (including gene of interest) is finally introduced into appropriate host cell or organism
- 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
- 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 desired trait encoded by the gene of interest
SIMPLER VERSION:
- gene of interest is excised / “cut” from the genomic dna of the source cell using restriction enzymes [/ mRNA can be treated w reverse transcriptase to produce short dna segments]
- plasmid (small circle of dna) from e. coli bacteria is selected to “host” the gene of interest, and this is known as a plasmid vector
- plasmid vector is cut with the same restriction enzyme that was used to cut out the gene of interest
- important as restriction enzymes leave behind “sticky ends” that can only stick to other “sticky ends” cut by the same restriction enzyme and not other restriction enzymes
- plasmid vector and cut gene of interest are mixed together; T4 ligase used to restore phosphodiester bonds between plasmid and gene of interest, re-circularising dna
- resultant plasmid is known as a recombinant plasmid, and is inserted into host bacteria cells via a process known as transformation
8
Q
what are the issues (risks and benefits) associated with gm crops?
A
- genetically modified organisms (GMOs) are used in agriculture to improve crop yields and reduce farming costs
- however the use of GM crops is a contentious issue, as economic benefits must be weighed against environmental risks
GM CROPS AND HUMAN HEALTH
- GM crops can be used to improve human nutritional standards, by incorporating genes for certain proteins, vitamin or vaccines
- additionally, GM crops can be manufactured that lack common natural allergens or toxins
- however, inclusion or removal of certain genes could trigger unexpected adverse health reactions in some individuals
- currently, not all foods with GM components are labelled, making informed decisions of use difficult for consumers
GM CROPS AND ECONOMIC CONSEQUENCES
- GM crops can include genes to enable them to grow in a wider range of environments (e.g. drought / frost / salinity resistance)
- GM crops can be manufactured to produce greater yields (crops can potentially grow larger and faster)
- can include genes which slow the rate of spoiling, leading to longer shelf lives for GM foods
- may possess resistance to certain viruses or produce toxins to pests (reducing need for use of pesticides)
- herbicide resistant crops can be used to allow for the easier killing of weeds (which compete with crops for soil nutrients)
- overall, an improved yield, reduction in farming costs and ability to farm more land will provide an economic benefit to farmers
- however, patent protection allows biotech companies to restrict the use of seeds and force farmers to pay high prices for use
GM CROPS AND ENVIRONMENTAL ISSUES
- ability to farm a wider range of environments with GM crops will potentially reduce the need for associated deforestation
- the generation of pest-resistant crops means that less chemical insecticides will be released into the environment
- however, GM crops could potentially reduce biodiversity in a region by competing with indigenous plant life
- furthermore, proteins or toxins produced by GM crops could negatively affect certain organisms within the ecosystem
- cross-pollination by GM crops could also result in the formation of herbicide-resistant weeds and grasses
- GM crops with pest toxins could also accelerate the evolution of resistant pest species
SUMMARY
BENEFITS:
- nutritional value of foods can be improved (introducing proteins, vitamins / vaccines)
- crops can be produced that lack known allergens
- crops can grow in arid conditions for better yield (introducing drought resistant genes)
- can produce herbicides to kill pests
- improve food supply / agriculture in poor countries (improved yields)
- reduces economic costs and carbon footprint (less need for land clearing and pesticide usage)
RISKS:
- new traits could cause adverse health reactions (new proteins could cause allergic response)
- removal of traits could have unknown effects
- crops may limit biodiversity of local environment (increased competition with native species)
- cross-pollination could lead to super weeds
- patents restrict farmers from accessing gm seeds (biotech companies hold monopoly over crop use)
- foods with gm components not labelled
- different governments may have conflicting regulatory usage
9
Q
what are the risks of Bt (Bacillus thuringiensis) crops on monarch butterflies?
A
- Bt corn is a genetically modified maize that incorporates an insecticide producing gene from the bacterium Bacillus thuringiensis
- insecticide lethal to certain types of larvae, particularly 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
- 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
- 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)
- larva were restricted in their diet (in the field, larva could feasibly avoid eating pollen dusted leaves)
- 2nd study was conducted comparing survivor rates of monarch butterflies based on proximity to Bt corn fields:
- no significant increase in mortality when monarch larva were placed in or near an actual Bt corn field
- concluded that exposure to Bt pollen poses no significant risk to monarch butterfly populations
10
Q
what are clones?
A
- 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
- mechanisms exist whereby sexually reproducing organisms can produce clones (e.g. identical twins)
- cloning multicellular organisms requires 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)
11
Q
what is somatic cell nuclear transfer (scnt)?
A
- method by which cloned embryos can be produced using differentiated adult cells
- (differentiated) somatic cells are removed from adult donor and cultured (cells are diploid and contain entire genome); nucleus of somatic cell is removed
- unfertilised egg is removed from a female adult and its haploid nucleus is removed to produce an enucleated egg cell
- enucleated egg cell fused with nucleus from the adult donor to make a diploid egg cell (with donor’s DNA)
- electric current delivered to stimulate the egg to divide and develop into an embryo
- embryo is then implanted into uterus of surrogate and will develop into a genetic clone of adult donor
12
Q
what are some natural animal cloning methods?
A
BINARY FISSION
- parent organism divides equally in 2, so as to produce 2 genetically identical daughter organisms
- occurs in planaria (flatworms) but is also common to bacteria and protists (e.g. euglena, amoeba)
BUDDING
- cells split off parent organism, generating a smaller daughter organism which eventually separates from 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 formed from unfertilised ova (via production of a diploid egg cell by the female)
- occurs in certain species of insect, fish, amphibians and reptiles
13
Q
what are some natural plant cloning methods?
A
- plants have capacity for vegetative propagation, where small pieces can be induced to grow independently
- adult plants possess meristematic tissue capable of cellular differentiation (totipotent)
- virtually all types of roots and shoots capable of vegetative propagation
- garlic and onion bulbs are modified plant leaves – all bulbs in a group are genetically identical
- underground stems (e.g. potato tubers) can form new plants which are genetically identical to parent plant
- certain plants can form horizontal stems called runners (or stolons) that grow roots and develop into clones
- some plants (mainly algae, mosses and ferns) can reproduce asexually by producing spores
- spores are also produced by certain types of bacteria and fungi
14
Q
what are some natural human cloning methods?
A
- 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
15
Q
what are the 2 methods of artificial cloning?
A
- embryo division
2. cloning using somatic cell nuclear transfer
