3.5 Genetic Modification Flashcards
What is PCR?
The polymerase chain reaction (PCR) is an artificial method of replicating DNA under laboratory conditions
What is PCR used for?
The PCR technique is used to amplify large quantities of a specific sequence of DNA from an initial minute sample
How many DNA molecules does each cycle of PCR produce?
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (230)
Where does PCR occur and why?
PCR occurs in a thermal cycler and uses variations in temperature to control the replication process
What is the first stage of PCR?
Denaturation – DNA sample is heated to separate it into two single strands (~95ºC for 1 min)
What is the second stage of PCR?
Annealing – DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min)
What is the third stage of PCR?
Elongation – A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min)
What is done with the resulting DNA copies from PCR?
Once large quantities of DNA have been created, other laboratory techniques are used to isolate and manipulate the sequences
What is gel electrophoresis?
Gel electrophoresis is a laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size
Where are samples placed in gel electrophoresis and what is done to them?
Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel
What fragments will move faster?
Smaller samples are less impeded by the gel matrix and hence will move faster through the gel
What does the differing size of the pieces in DNA mean in electrophoresis?
This causes samples of different sizes to separate as they travel at different speeds
Is the process for separating DNA and proteins the same?
While both DNA and proteins are separated according to the same basic process, differences exist between the two protocols
What cuts DNA into pieces?
DNA may be cut into fragments using restriction endonuclease – different DNA samples will generate different fragment lengths
Why do DNA fragments separate?
Fragments separate because DNA is negatively charged due to the presence of a phosphate group (PO43–) on each nucleotide
What gel are DNA fragments placed in?
DNA samples are placed into an agarose gel and fragment size calculated by comparing against known industry standards
How can specific DNA sequences be identified in gel electrophoresis?
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)
What is the basis of protein separation by gel electrophoresis?
Proteins may be folded into a variety of shapes (affecting size) and have positive and negative regions (no clear charge)
What are proteins first treated with? gel electrophoresis
Proteins must first be treated with an anionic detergent (SDS) in order to linearise and impart a uniform negative charge
What are protein samples placed into? gel electrophoresis
Protein samples are placed into a polyacrylamide gel and sizes compared against known industry standards
How are target proteins identified via gel electrophoresis?
Separated proteins are transferred to a membrane and then target proteins are identified by staining with specific monoclonal antibodies (Western blotting)
What is DNA profiling?
DNA profiling is a technique by which individuals can be identified and compared via their respective DNA profiles
What is within an individual’s genome that is used for DNA profiling?
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)
WHy are short tandem repeats used for DNA profiling?
As individuals will likely have different numbers of repeats at a given satellite DNA locus, they will generate unique DNA profiles
When is DNA profiling commonly used?
DNA profiling is commonly used in criminal investigations (forensics) and to settle paternity disputes
When is DNA profiling commonly used?
DNA profiling is commonly used in criminal investigations (forensics) and to settle paternity disputes
What is the first step for DNA profiling?
A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR
What is done with the collected sample? DNA Profiling
Satellite DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments
What will differ in DNA profiling?
Fragment length will differ between individuals due to the variable length of their short tandem repeats
What separates the fragments in DNA profiling?
The fragments are separated using gel electrophoresis and the resulting profiles are compared
What must the DNA profile show if a conviction is to occur, DNA Profiling?
Suspects should be a complete match with the DNA sample taken from the crime scene if a conviction is to occur
What does the number of loci used to generate a unique profile depend on?
The number of loci used to generate a unique profile depends on the size of the population being compared
How can DNA profiling be used to determine paternity?
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
How does a gene determine a particular trait?
A gene determines a particular trait by encoding for a specific polypeptide in a given organism
What does the universality of the genetic code allow for in terms of genetic modification?
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 created?
The transfer of genes between species is called gene modification, and the new organism created is called a transgenic
WHat are the 4 key 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 transgenic construct
1.1. How can DNA be isolated from cells? GT (gene transfer)
DNA can be isolated from cells by centrifugation – whereby heavier components such as nuclei are separated
1.2. What is done to the gene of interest? GT.
The gene of interest can then be specifically amplified via the polymerase chain reaction (PCR)
1.3 What is an alternate way of generating the gene sequences? GT
Gene sequences can also be generated from mRNA using reverse transcriptase – these DNA sequences (cDNA) lack introns
1.4 What is a vector?
A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell
1.5 What is a common vector in gene transfer?
Bacterial plasmids are commonly used as vectors because they are capable of autonomous self-replication and expression
1.6 What MAY be done to bacterial plasmids? GT
These plasmids may be modified for further functionality (e.g. selection markers, reporter genes, inducible expression promoters)
1.7 What are other types of vectors used in gene transfer?
Other types of vectors include modified viruses and artificial chromosomes
2.1 What must be done to incorporate the gene of interest into the vector? GT
In order to incorporate a gene of interest into a vector, both must be cut with restriction enzymes at specific recognition sites
2.2 What do restriction enzymes do? GT
Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs)
2.3 How will the vector and gene be joined? GT
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
3.1.Where is the gene of interest inserted? GT
The gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases
3.2 Why do the gene of interest and plasmid join together? GT
This occurs because the sticky ends of the gene and vector overlap via complementary base pairing
3.3 What is a recombinant construct and how is it formed? GT
The gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct
3.4 What enzymes joins the desired gene and plasmid together? GT
DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond
4.1 What is done with the recombinant construct? GT
The recombinant construct (including the gene of interest) is finally introduced into an appropriate host cell or organism
4.2 How can the process of introducing the recombinant construct into the organism be achieved?
This process can be achieved in a variety of ways and is called transfection (for eukaryotes) or transformation (for prokaryotes)
4.3 What is a common way to identify which cells have successfully incorporated the human gene?
Antibiotic selection is commonly used in order to identify which cells have successfully incorporated the recombinant construct
4.4 How is antibiotic selection used to identify genes which have incorporated the human gene?
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
Why are GMO’s used in agriculture?
Genetically modified organisms (GMOs) are used in agriculture to improve crop yields and reduce farming costs
What may be a general con of GMOs in agriculture?
However the use of GM crops is a contentious issue, as economic benefits must be weighed against environmental risks
WHat are 2 pros of GMOs in terms of 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
What are 2 cons of GMOs on human health?
However, the 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
What are economic consequences of GMOs? 5!
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)
GM crops can include genes which slow the rate of spoiling, leading to longer shelf lives for GM foods
GM crops may possess resistance to certain viruses or produce toxins to pests (reducing need for the use of pesticides)
Herbicide resistant crops can be used to allow for the easier killing of weeds (which compete with crops for soil nutrients)
What is the general economic pro and con of GMOS?
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
What are environmental issues of GMOs? 5
The ability to farm a wider range of environments with GM crops will potentially reduce the need for associated deforestation
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
What is an environmental pro of GMOs?
Also, the generation of pest-resistant crops means that less chemical insecticides will be released into the environment
What is Bt corn?
t corn is a genetically modified maize that incorporates an insecticide producing gene from the bacterium Bacillus thuringiensis
What is the insecticide of Bt corn lethal to?
This insecticide is lethal to certain types of larvae, particularly the European corn borer which would otherwise eat the crop
What may the spread of Bt corn affect?
Concerns have been raised that the spread of Bt corn may also be impacting the survival rates of monarch butterflies
How does Bt corn affect monarch butterfly larvae?
While monarch butterfly larva feed exclusively on milkweed, wind-borne pollen from Bt corn may dust nearby milkweeds
What did a preliminary study do to investigate 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)
What was compared in this preliminary study?
Growth and mortality rates were compared against caterpillars fed on non-dusted leaves or leaves dusted with non-GM pollen
What were the results of the preliminary study?
Caterpillars exposed to Bt pollen were found to have eaten less, grew more slowly and exhibited higher mortality rates
What is a criticisim of this preliminary study?
Some scientists suggested that these results may lack validity as they do not accurately reflect natural conditions
Name two ways in which this preliminary study lacked validity.
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)
What did a second study investigate? (Bt & Monarchs)
A second study was conducted comparing the survivor rates of monarch butterflies based on proximity to Bt corn fields
What were the two findings of the second study?
There was no significant increase in mortality when monarch larva were placed in or near an actual Bt corn field
From this it was concluded that exposure to Bt pollen poses no significant risk to monarch butterfly populations
What are clones?
Clones are groups of genetically identical organisms or a group of cells derived from a single original parent cell
What organisms produce clones?
Organisms that reproduce asexually will produce genetically identical clones
Can sexually reproducing organisms generate clones?
YES
Additionally, mechanisms exist whereby sexually reproducing organisms can produce clones (e.g. identical twins)
What does the cloning of multicellular organisms require?
Cloning multicellular organisms requires the production of stem cells (differentiated cells cannot form other cell types)
How can stem cells be artificially generated?
Stem cells can be artificially generated from adult tissue using a process called somatic cell nuclear transfer (SCNT)
What is somatic cell nuclear transfer?
Somatic cell nuclear transfer is a method by which cloned embryos can be produced using differentiated adult cells
What is removed in SCNT?
Somatic cells are removed from the adult donor and cultured (these cells are diploid and contain the entire genome)
What is taken from the female adult (SCNT)?
An unfertilised egg is removed from a female adult and its haploid nucleus is removed to produce an enucleated egg cell
What is the egg cell fused with? (SCNT)
The enucleated egg cell is fused with the nucleus from the adult donor to make a diploid egg cell (with the donor’s DNA)
What then stimulates the egg cell (SCNT)?
An electric current is then delivered to stimulate the egg to divide and develop into an embryo
Where is the egg then implanted SCNT?
The embryo is then implanted into the uterus of a surrogate and will develop into a genetic clone of the adult donor
Do many species possess natural mechanisms of cloning?
YES
Many species can reproduce asexually and hence possess natural methods of cloning
What species can reproduced asexually to produce genetic clones?
All bacteria, the majority of fungi and many species of protists reproduce asexually to produce genetic clones
In what ways do plants reproduce?
While most plants reproduce sexually, they also possess methods of asexual reproduction (vegetative propagation)
Can animals reproduce asexually?
Certain animal species can also reproduce asexually, via a variety of different mechanisms
What are 4 animal cloning methods?
binary fission
budding
fragmentation
parthenogenesis
What does binary fission involve?
The parent organism divides equally in two, so as to produce two genetically identical daughter organisms
In what organisms does binary fission occur?
This method of cloning occurs in Planaria (flatworms) but is also common to bacteria and protists (e.g. euglena, amoeba)
What does budding involve?
Cells split off the parent organism, generating a smaller daughter organism which eventually separates from the parent
In what organisms does budding occur?
This method of cloning occurs in Hydra but is also common to many species of yeast
WHat does fragmentation involve?
New organisms grow from a separated fragment of the parent organism
In what organisms does fragmentation occur?
This method of cloning is common to starfish and certain species of annelid worms
What does parthenogenesis involve?
Embryos are formed from unfertilised ova (via the production of a diploid egg cells by the female)
In what organisms does parthenogenesis occur?
This method of cloning occurs in certain species of insect, fish, amphibians and reptiles
In what way can plants produce clones naturally?
Plants have the capacity for vegetative propagation, whereby small pieces can be induced to grow independently
Why is vegetative propagation possible in plants?
This is because adult plants possess meristematic tissue capable of cellular differentiation (totipotent)
What plants are capable of vegetative propagation?
Virtually all types of roots and shoots are capable of vegetative propagation
What are garlic and onion bulbs?
Garlic and onion bulbs are modified plant leaves – all the bulbs in a group are genetically identical
What stems can form new plants?
Underground stems (e.g. potato tubers) can form new plants which are genetically identical to the parent plant
What other types of stems can certain plants form?
Certain plants can form horizontal stems called runners (or stolons) that grow roots and develop into clones
Apart from vegetative propagation, in what ways can some plants reproduce asexually?
Some plants (mainly algae, mosses and ferns) can reproduce asexually by producing spores
Spores are also produced by certain types of bacteria and fungi
What natural cloning method is present in humans?
identical twins
How are identical twins created?
Identical twins (monozygotic) are created when a fertilised egg (zygote) splits into two identical cells, each forming an embryo
How are non-identical twins formed?
Non-identical twins (dizygotic) are created when an unfertilised egg splits into two cells and each is fertilised by a different sperm
How genetically similar will identical and non-identical twins be?
Identical twins will be clones of one another (genetically identical), while non-identical twins will share 50% of the same DNA
What do embryonic cells retain at a very early stage?
At a very early stage, embryonic cells retain pluripotency (meaning they can divide and become any type of tissue)
What will embryonic cells form?
These cells will differentiate to form all the different tissues comprising the organism
What will happen if these embryonic cells are separated artificially?
If these embryonic cells are separated artificially in the laboratory, each group of cells will form cloned organisms
Can the separation of embryonic cells occur naturally?
This separation of embryonic cells can also occur naturally to give rise to identical (monozygotic) twins
When should the separation if embryonic cells occur/
The separation of embryonic cells has to happen early in the developmental cycle, ideally around the 8 cell stage (morula)
What is done with the separated group of cells?
The separated groups of cells are then implanted into the uterus of a surrogate to develop into genetically identical clones
What is the method of separating embryonic stem cells limited by?
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
What is a more reliable method of cloning?
A second and more reliable method of artificial cloning involves somatic cell nuclear transfer (SCNT)
What does SCNT involve? general
This involves replacing the haploid nucleus of an unfertilised egg with a diploid nucleus from an adult donor
What is the advantage of this technique?
The advantage of this technique is that it is known what traits the clones will develop (they are genetically identical to the donor)
What are the two main purposes of using differentiated cells to generate cloned embryos?
reproductive and therapeutic cloning
What is reproductive cloning?
Reproductive cloning: If the embryo is implanted into the uterus of a surrogate, a new cloned organism will develop
What is therapeutic cloning?
Therapeutic cloning: Embryonic cells can be induced to differentiate to create specific tissues or organs for transplantation
What is a stem cutting?
A stem cutting is a separated portion of plant stem that can regrow into a new independent clone via vegetative propagation
What do all stems possess?
All stems possess nodes, from which a leaf, branch or aerial root may grow – the region between nodes are called internodes
Where are stem cuttings typically placed?
Stem cuttings are typically placed in soil with the lower nodes covered and the upper nodes exposed
What is stem cutting commonly used for?
Stem cutting is a common method employed to rapidly propagate plant species (including sugar cane, grapes and roses)
What are the 7 factors influencing the successful rooting of a stem cutting?
cutting position
length of cutting
growth medium
use and concentration of hormones
temperature conditions
availability of water
other environmental conditions
How does cutting position influence the success of rooting of a stem cutting?
(whether cutting occurs above or below a node, as well as the relative proximity of the cut to the node
How does length of cutting influence the success of rooting of a stem cutting?
Length of cutting (including how many nodes remain on the cutting)
How does growth medium influence the success of rooting of a stem cutting?
Growth medium (whether left in soil, water, potting mix, compost or open air)
How does use and concentration of hormones influence the success of rooting of a stem cutting?
The use and concentration of growth hormones (e.g. IAA, IBA and NAA promote the formation of adventitious roots)
How do temperature conditions influence the success of rooting of a stem cutting?
Temperature conditions (most cuttings grow optimally at temperatures common to spring and summer)
How does the availability of water influence the success of rooting of a stem cutting?
Availability of water (either in the form of ground water or humidity)
What environmental factors can affect the successful rooting of a stem cutting?
including pH of the soil and light exposure
