SL Genetics : Genetic Modification and Biotechnology (3.5)

Define PCR

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 amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (2³⁰)

Explain the stages of PCR

PCR occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:

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)

Once large quantities of DNA have been created, other laboratory techniques are used to isolate and manipulate the sequences

Outline the mechanism of Gel Electrophoresis

Gel electrophoresis is 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

Explain (in detail) the stages of Gel Electrophoresis

DNA Separation

• 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 (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)

Explain what is used for DNA Profiling

• 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

Define DNA Profiling

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 

Explain how DNA Profiling is used in Forensic Investigations and Paternity Tests

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

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

Explain the process of Gene Transfer for Genetic Modification

Step 1:  Isolating gene and vector

• 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

Step 2:  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

Step 3:  Ligation of Vector and Insert

• 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

Step 4:  Selection and Expression

• 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
  

Outline the relationship between Human Health and GMOs

• 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, 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

Outline Gene Transfer

Because the genetic code is (almost) 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, and the new organism created is called a transgenic

Outline the economic consequences of GM crops

• 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)

• 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

Outline the environmental issues surrounding GM crops

• The ability to farm a wider range of environments with GM crops will potentially reduce the need for associated deforestation

• Also, 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

Summarize the risks and benefits of GM Crops

Define a ‘Clone’

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)

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)

Explain the Production of cloned embryos produced by somatic cell nuclear transfer

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 unfertilised 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 

Provide examples of Natural Cloning of Animals

Animal Cloning Methods

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)

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

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

Parthenogenesis

• Embryos are formed from unfertilised ova (via the production of a diploid egg cells by the female)

• This method of cloning occurs in certain species of insect, fish, amphibians and reptiles

Provide examples of Natural Cloning of Plants

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) 

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

Some plants (mainly algae, mosses and ferns) can reproduce asexually by producing spores

• Spores are also produced by certain types of bacteria and fungi

Provide examples of Natural Cloning of Humans

Even human beings are capable of creating genetic clones through natural means

• 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

Explain the process of Artificial Cloning

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

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

Describe another method of artificial cloning : 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 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 (they are genetically identical to the donor)

What is the purpose of SCNT

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

Define a Stem Cutting

A stem cutting is a separated portion of plant stem that can regrow into a new independent clone via vegetative propagation

• 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 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 influence successful 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 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 ground water or humidity)

• Other environmental conditions (including pH of the soil and light exposure)