3.5 Genetic modification and biotechnology

Question 1

What is PCR and what is it used for?

Answer 1

• 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)

Question 2

Describe the stages of PCR

Answer 2

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)

Question 3

What happens once large quantities of DNA have been created through PCR?

Answer 3

Other laboratory techniques are used to isolate and manipulate the sequences.

Question 4

Diagram showing an overview of the PCR cycle

Answer 4

Question 5

What is gel electrophoresis?

Answer 5

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

Question 6

Give an overview of how gel electrophoresis works

Answer 6

• 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

Question 7

Diagram of the gel electrophoresis apparatus

Answer 7

Question 8

Are DNA and protein separations the same?

Answer 8

While both DNA and proteins are separated according to the same basic process, differences exist between the two protocols

Question 9

Explain how DNA separation using gel electrophoresis works

Answer 9

• 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)

Question 10

Diagram of agarose gel electrophoresis (DNA)

Answer 10

Question 11

Explain how protein separation using gel electrophoresis works

Answer 11

-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)

Question 12

Diagram of polyacrylamide gel electrophoresis (proteins)

Answer 12

Question 13

What is DNA profiling and how does it work?

Answer 13

-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

Question 14

Diagram of comparative STR lengths at two specific loci

Answer 14

Question 15

What are the uses of DNA profiling?

Answer 15

For criminal investigations (forensics) and to settle paternity disputes (among other uses)

Question 16

Describe the procedure of DNA profiling

Answer 16

-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

Question 17

How can DNA profiling be used in forensic investigations?

Answer 17

-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

Question 18

How can DNA profiling be used in paternity testing?

Answer 18

-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

Question 19

Diagram of the DNA profiles of DNA found at a crime scene

Answer 19

Question 20

In this DNA profile, who is the biological father?

Answer 20

Father 2

Question 21

Key points about analyzing DNA evidence

Answer 21

-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.

Question 22

What conclusion can be made from the following DNA profiling evidence?

Answer 22

-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.

Question 23

What does a gene determine and how?

Answer 23

It determines a particular trait by encoding a specific polypeptide in a given organism.

Question 24

Why is gene transfer possible?

Answer 24

Because the genetic code is (almost) universal, an organism can potentially express a new trait if the appropriate gene is introduced into its genome.

Question 25

What is genetic modification and what is a transgenic organism?

Answer 25

The transfer of genes between species is called gene modification, and the new organism created is called a transgenic.

Question 26

Diagram showing bacteria producing human insulin (Bioninja)

Answer 26

Question 27

Diagram showing a genetic modification of a bacterium to produce human insulin

Answer 27

Question 28

What are the steps of gene transfer?

Answer 28

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 the transgenic construct

Question 29

Explain step 1 of gene transfer: Isolating gene and vector (by PCR)

Answer 29

• 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

Question 30

Diagram showing the common features of a typical plasmid vector

Answer 30

Question 31

Explain step 2 of gene transfer: digestion with restriction enzymes

Answer 31

• 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

Question 32

Diagram showing ‘sticky end’ vs. ‘blunt end’ restriction enzymes

Answer 32

Question 33

Explain step 3 of gene transfer: Ligation of gene and vector (by DNA ligase)

Answer 33

-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

Question 34

Diagram of the formation of a recombinant construct

Answer 34

Question 35

Explain step 4 of gene transfer: selection and expression of the transgenic construct

Answer 35

-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

Question 36

Diagram showing the isolation of transgenic cells via antibiotic selection

Answer 36

Question 37

Why are plasmids useful in genetic modification of bacteria?

Answer 37

They are easily transferred between bacterial cells and are small enough to manipulate easily.

Question 38

What is used to seal a target gene into a bacterial plasmid?

Answer 38

DNA ligase

Question 39

What separates genetic modification from earlier methods of manipulating offspring such as selective breeding?

Answer 39

Genetic modification transfers genes between different species.

Question 40

Describe the results of large-scale studies about GM crops

Answer 40

-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.

Question 41

Benefits of GMO crops

Answer 41

-Introduction of new positive traits to the crop

-Economic advantages

-Environmental advantages

Question 42

Risks of GMO crops

Answer 42

-Ecosystem damage

-Increasing monoculture

-Corporate control over food supply

-Human health concerns

Question 43

Explain how the introduction of new positive traits to the crop is a benefit of GMOs and give an example

Answer 43

-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.

Question 44

Explain how economic advantages are a benefit of GMOs and give an example

Answer 44

-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.

Question 45

Explain how environmental advantages are a benefit of GMOs

Answer 45

-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.)

Question 46

Explain how ecosystem damage is a risk of GMOs and give an example

Answer 46

-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.

Question 47

Explain how increasing monoculture is a risk of GMOs and give an example

Answer 47

-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.)

Question 48

Explain how corporate control over food supply is a risk of GMOs and give an example

Answer 48

-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.

Question 49

Explain how human health concerns are a risk of GMOs and give an example

Answer 49

-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.

Question 50

It is important to remember that ___ being transferred during genetic modification matters greatly when considering possible impacts.

Answer 50

The nature of the genes

Question 51

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?

Answer 51

-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.

Question 52

What is Bt corn and how can it be harmful?

Answer 52

-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

Question 53

What concerns have been raised about the effect of Bt corn?

Answer 53

-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

Question 54

Diagram showing the relationship between Bt corn and the monarch butterfly

Answer 54

Question 55

Describe the data that investigated the effect of Bt corn on monarch butterflies

Answer 55

-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

Question 56

Graph showing the effect of Bt pollen on survival rates of monarch butterflies (laboratory conditions)

Answer 56

Question 57

Why have some scientists questioned the 1999 preliminary study about the effects of Bt corn?

Answer 57

-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)

Question 58

Describe the second study on the effect of Bt corn

Answer 58

-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

Question 59

Diagram showing the effect of Bt pollen on survival rates in monarch butterflies (field conditions)

Answer 59

Question 60

What is a stem cutting?

Answer 60

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

Question 61

What are the features of stems and how are stem cuttings used?

Answer 61

-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)

Question 62

What factors will influence the success of rooting of a stem cutting?

Answer 62

-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)

Question 63

Diagram of germination of stem cuttings

Answer 63

Question 64

What are clones and how do they come about?

Answer 64

-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)

Question 65

What does cloning of multicellular organisms require?

Answer 65

-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)

Question 66

Explain how somatic cell nuclear transfer can produce cloned embryos

Answer 66

-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

Question 67

Diagram showing somatic cell nuclear transfer

Answer 67

Question 68

Give an overview of natural methods of cloning

Answer 68

-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

Question 69

Give examples of animal cloning methods

Answer 69

-Binary fission

-Budding

-Fragmentation

-Parthenogenesis

Question 70

Explain binary fission

Answer 70

-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)

Question 71

Explain budding

Answer 71

-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

Question 72

Explain fragmentation

Answer 72

-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

Question 73

Explain parthenogenesis

Answer 73

-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

Question 74

Diagram showing the methods of animal cloning

Answer 74

Question 75

Explain how plants can clone themselves

Answer 75

-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)

Question 76

Give examples of vegetative propagation

Answer 76

-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

Question 77

Describe asexual reproduction in plants

Answer 77

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

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

Question 78

Diagram of vegetative propagation via plant stolons

Answer 78

Question 79

Explain natural cloning in humans

Answer 79

-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

Question 80

Diagram of monozygotic vs. dizygotic twins

Answer 80

Question 81

Explain how animals can be cloned at the embryo stage

Answer 81

-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

Question 82

What is a limitation of the separation of embryonic cells artificially

Answer 82

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.

Question 83

Diagram of cloning via embryonic division

Answer 83

Question 84

Explain somatic cell nuclear transfer

Answer 84

-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)

Question 85

What is somatic cell nuclear transfer (SCNT) used for?

Answer 85

-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

Question 86

Diagram comparing reproductive and therapeutic cloning

Answer 86

Question 87

What happens to the unfertilized egg used in somatic-cell nuclear transfer?

Answer 87

Its nucleus is replaced by the nucleus of the differentiated body cell.