Topic 3.5 - Genetic Modification and Biotechnology

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 how binary fission create animal clones.

DNA is negatively charged and different DNA fragments will generate different fragment lengths

What two characteristics of DNA fragments allow successful DNA profiling via gel electrophoresis?

The use and safety of GMO crops is much debated. Maize has been modified to contain a gene from a bacterium (Bacillus thuringiensis). When ingested, the protein product of this gene (Bt toxin) kills a small range of insects. Corn borers cause great damage to maize crops and are killed by Bt toxin. Benefits: Reduced use of chemical insecticides. Less money spent on insecticides. Less damage to wildlife.Farmers are not handling toxic insecticides. Improved yield, as less damage. Improved grain quality. Due to less damage by borers, there is less fungus growth on the corn. Some fungal toxins are carcinogenic. Risks: Potential risk to non-target insects, such as Monarch butterflies. When feeding in nearby regions, butterflies could ingest pollen blown from Bt maize plants. Insects may become resistant to Bt toxin. Transfer of the Bt gene to wild relatives of corn by cross pollination. Thus affecting insects feeding on them.

Assess the benefits and risks of genetically modified crops using Bt maize as an example.

an organism or cell, or group of organisms or cells, produced asexually from one ancestor or stock, to which they are genetically identical.

Define a clone

A laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size

Define gel electrophoresis

Genetic modification is the process of altering the genetic makeup of an organism.

Define genetic modification

The gene responsible for insulin production is extracted from a human cell. It is spliced into a plasmid vector (for autonomous replication and expression) before being inserted into a bacterial cell The transgenic bacteria (typically E. coli) are then selected and cultured in a fermentation tank (to increase bacterial numbers) The bacteria now produce human insulin, which is harvested, purified and packaged for human use (i.e. by diabetics)

Describe how can gene transfer been used to help make commercially sold insulin.

Taking cuttings from stems is one way plants (such as Fuchsia) can be propagated. Take 5-10 cm cuttings from the tips of non-flowering shoots, cutting just below the leaf nodes. Remove the bottom leaves. Insert the cutting (up to the lowest leaves) into a pot containing damp compost mixed with vermiculite. Cover the pot containing the cutting with a clear plastic bag to create high humidity (to reduce transpiration) and keep in a light, warm place. Possible dependent variables: If roots form. Number of roots formed. Possible independent variables: Applying plant hormones to the cut stem. Temperature Humidity Variables not selected as the independent variable must be controlled.

Design an experiment to test a factor that affects the formation of roots on stem-cuttings.

Genes from Bt have been inserted into maize so GM plants can produce an insecticidal toxin and therefore be resistant to pests, e.g. European Corn Borer.

Explain an example of gene transfer utilizing a plasmid from a bacterium.

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

Explain how Bt corn was produced. State the concern about Bt corn with monarch butterflies.

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 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)

Explain how DNA is separated in Gel Electrophoresis

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

Explain how parthenogenesis create animal clones.

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 how protein is separated in Gel Electrophoresis

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 how splitting or fragmentation create animal clones.

Forensic testing - Suspects should be a complete match with the DNA sample taken from the crime scene if a conviction is to occur parental testing - Children inherit half their chromosomes from each parent and thus should possess a combination of parental fragments

Explain some applications of DNA profiling.

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

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

Environmental: 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 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, 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 Economic: 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

GM crops have been assessed on their possible environmental, health and economic benefits and risks. Provide a claimed benefit and risk for each one.

Karyotyping looks at the size, shape and number of your chromosomes, which are able to distinguish sex, down syndrome etc. While DNA profiling looks at samples DNA based on their size to match unknown DNA to a person.

How is karyotyping different from DNA profiling?

Allows replication of DNA, so if there is only a very small sample of DNA, PCR will be able to replicate the DNA, so it is identical and then there will be more DNA.

Identify benefits of performing PCR.

Reproductive cloning

If the embryo is implanted into the uterus of a surrogate, a new cloned organism will develop

DNA profiling or genetic fingerprinting compares DNA from two or more individuals. There are several known regions of the genome that vary between people. In these regions there are many repeated units of short DNA sequences (called short tandem repeats or STRs). The more repeats, the longer the region. Method of DNA profiling: Collect DNA samples from individuals. Contamination of samples must be avoided. Use PCR to amplify several different variable regions of the genome. Separate DNA fragments by gel electrophoresis. The pattern of bands revealed by gel electrophoresis will be unique to individuals (except identical twins). The two main uses of DNA profiling are: Placing a suspect to a crime scene (forensics) and Paternity testing.

Outline DNA profiling.

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

Outline how the structure of gel electrophoresis helps it achieve its purpose.

DNA strands are separated by heating to 94 °C. The temperature is lowered to about 45 °C to allow primers to bind to the single strands of the DNA. The temperature is raised to 72 °C to provide the optimum temperature for Taq DNA polymerase to form new DNA strands extending from the primers.

Outline the method of polymerase chain reaction (PCR).

Isolate the gene of interest (e.g. the insulin gene). Cut human DNA with restriction endonuclease. Or use reverse transcriptase to make a DNA copy of insulin mRNA (extracted from pancreas beta cells). Cut a plasmid with the same restriction endonuclease used to cut the human DNA. If reverse transcriptase was used, sticky ends must be added. Mix cut plasmids and the insulin gene with DNA ligase to join the molecules. The plasmid is now called a recombinant DNA molecule as it contains DNA from two species. The recombinant plasmids are inserted into bacteria (host) cells. The genetically modified bacteria are grown in large fermenters and insulin can be extracted.

Outline the process of gene transfer to create genetically modified bacteria.

Somatic cell nuclear transfer allows adult animals to be cloned from a differentiated body cell. This method was used to clone Dolly, the sheep. An udder cell (a differentiated cell) was removed from an adult sheep. This sheep is the donor. A second sheep provides an unfertilized egg. The nucleus is removed from the egg. The udder cell and the egg without the nucleus are fused. Electrical pulses are given to facilitate the fusion. The fused cell is grown in culture until it reaches the 16 cell stage. It is then implanted into a third sheep, who will be the surrogate mother. The surrogate mother gives birth to the cloned lamb. The lamb is genetically identical to the donor sheep.

Outline the process of somatic cell nuclear transfer.

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

Outline the stages of DNA profiling.

Gel electrophoresis is used to separate DNA fragments or proteins according to their molecular size. The gel has a porous structure, through which DNA fragments or proteins can pass. An electric current is used to move molecules through the gel. DNA is negatively charged so it will be repelled by the negative electrode and attracted to the positive electrode. Proteins are treated to make them negatively charged. Larger molecules will pass more slowly through the pores of the gel than smaller ones. Hence, smaller molecules will travel further through the gel than larger molecules in a given time.

Outline the technique of gel electrophoresis.

Binary Fission, Budding, Fragmentation, Parthenogenesis

State four natural ways plants and animal species clone

PCR is necessary for DNA profiling because it is used to amplify the amount of the DNA sample that is collected.

State why PCR is necessary for DNA profiling?

Therapeutic cloning

The cloning of human cells by nuclear transplantation for therapeutic purposes, such as the generation of embryonic stem cells to treat disease.

Since the genetic code is universal genes can be transferred between species and still allow for the same polypeptide to be translated.

The genetic code is universal. Explain how that allows gene transfer to be possible.

a. A vector is used to transfer genes between species. For example, plasmids. b. There are many different restriction endonuclease enzymes that cut DNA at specific recognition sequences. Overhangs are created when the DNA molecule is cut - these are called sticky ends. c. When sticky ends join back together (due to complementary base pairing), DNA ligase is needed to join the sugar-phosphate backbone back together. d. Can synthesize a DNA copy from a mRNA molecule. e. The organism that the vector is transferred into. Typically bacteria are host organisms.

The process of gene transfer creates genetically modified organisms. Outline the role of the following in this process: a. Vector b. Restriction endonuclease c. DNA ligase d. Reverse transcriptase e. Host organism

Genetically modified crops. Genetically modified crops (GM crops) are plants used in agriculture, the DNA of which has been modified using genetic engineering methods. In most cases, the aim is to introduce a new trait to the plant which does not occur naturally in the species.

What are GMOs or GM crops?

A clone is an organism derived from a single parent cell and is genetically identical to the parent cell. Different organisms cloned from the same parent cell will also be genetically identical. This can occur naturally or artificially. Natural clones: Asexual reproduction occurs in plants and animal species. For examples, plants can form tubers, bulbs, or runners (vegetative propagation). Animals, such as hydra, bud off new individuals. Artificial clones: With plants, cuttings are taken from the stem and rooted. Animals: Embryos can be isolated and broken up. Each smaller group of cells will produce an individual. By manipulation of reproductive cells, a process called somatic-cell nuclear transfer.

What is a clone and how are they produced?

Restriction enzymes cut at specific sequences so the same restriction enzyme must be used because it will produce fragments with the same complementary sticky ends, making it possible for bonds to form between them.

Why must the same restriction enzyme be used on both samples of DNA of the organisms involved?

Transgenic

organism whose genome has been altered to contain one or more genes from another organism or species