3.5 Genetic modification and biotechnology

Question 1

What is the use of a PCR?

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

Question 2

3 stages of a 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?

Answer 3

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

Question 4

What is the use of a gel electrophoresis?

Answer 4

Gel electrophoresis is a laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size

Question 5

How does a gel electrophoresis work?

Answer 5

• 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 6

What can gel electrophoresis be used on?

Answer 6

Both DNA and proteins. They are both separated using the same basic process, but thee are differences between the two protocols

Question 7

How is DNA separated using gel electrophoresis?

Answer 7

• 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)

Question 8

How are proteins seperated using a gel electrophoresis?

Answer 8

• 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)

Question 9

What is DNA profiling and what does it show?

Answer 9

DNA profiling is a technique by which individuals can be identified and compared via their respective DNA profiles

Question 10

What generates unique DNA profiles?

Answer 10

• Satellite DNA (long stretches of DNA made up of repeating elements called short tandem repeats -STRs) are part of the non-coding regions of an individuals genome and each individual is likely to have different numbers of repeats at a given satellite DNA locus, and that generates unique DNA profiles.

Question 11

When is DNA profiling used? What is the procedure used?

Answer 11

DNA profiling is commonly used in criminal investigations (forensics) and to settle paternity disputes

The procedure involved is common for both:

• 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

Question 12

When using DNA profiling in forensic investigations, what can you expect?

Answer 12

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 13

When using DNA profiling in paternity testing, what can you expect?

Answer 13

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 14

What is a transgenic organism?

Answer 14

A new organism created e from the transfer of genes between species (gene modification)

Question 15

What can we do about the fact that the genetic code is (almost) universal? an example?

Answer 15

We can introduce an appropriate gene to an organisms genome to potentially express a new trait

Insert human gene into a bacteria plasmid to produce human insulin

Question 16

How is insulin produced through bacteria?

Answer 16

1. The DNA for insulin is first isolated
2. A plasmid made by DNA is removed from a bacterial cell
3. A restriction enzyme cuts the plasmid DNA, leaving sticky ends (single stranded sections that have complementary base sequences so can be used to link together pieces of DNA, by hydrogen bonding between the bases)
4. The insulin gene, with complementary sticky ends is added
5. DNA ligase enzyme splices together (joins) the plasmid DNA and Insulin DNA by making sugar-phosphate bonds between nucleotides to seal the sticky ends together
6. The plasmid (now genetically modified) is inserted back into the bacterium
7. The bacterium host cell, divides and produces copies of the plasmid
8. The bacterium makes human insulin using the gene in the plasmid
9. The insulin is extracted from the bacterial culture

Question 17

What do restriction enzymes do?

Answer 17

• Also known as endonucleases
• Restriction enzymes (like DNA scissors)
• Binds to specific DNA sequences.
• Cut the DNA at this specific place
• Leave ‘sticky ends’ of DNA single strands

(There are many types of restriction enzymes)

Question 18

What is DNA ligase?

Answer 18

• An enzyme that joins together DNA strands
• Connect the sticky ends together
• Splices the new gene into the DNA

Question 19

Why is reverse transcriptase used? and what is it?

Answer 19

Reverse transcriptase is an enzyme that makes DNA copies of RNA molecules called cDNA. It can be used to make the DNA needed for gene transfer from messenger RNA.

Question 20

What are two examples of GMOs?

Answer 20

Golden Rice: Rice modified with daffodil genes to have more beta-carotene, which the body converts to Vitamin A

Bt Corn: Corn modified with a bacterial insecticide gene so that it produces insect toxins within its cells, protecting it from pest species

Question 21

What are environmental and health, benefits of GM crops?

Answer 21

Environmental: Pest resistant crop varieties can be produced by transferring a gene for making a toxin to the plants. Less insecticide then has to be sprayed on to the crop so fewer bees and other beneficial insects are harmed. (Bt Corn)

Health: The nutritional value of crops can be improved, for example by increasing the vitamin content (Golden Rice)

Question 22

What are the health and environmental risk of GMOs?

Answer 22

Health: Proteins produced by transcription and translation of transferred genes could be toxic or cause allergic reactions in humans or livestock that eat GM crops (Golden rice?)

Environmental: Non-target organisms could be affected by toxins that are intended to control pests in GM crop plants (Bt corn)

Question 23

What is the relationship between Bt crops and Monarch butterflies?

Answer 23

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

Question 24

What are clones? What reproduction must they undergo?

Answer 24

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 25

What does cloning of multicellular organisms require? How can stem cells be artificially generated?

Answer 25

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)

Question 26

What is somatic cell nuclear transfer (SCNT)? What is the process?

Answer 26

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

Question 27

How does natural cloning occur? Who does it?

Answer 27

When species reproduce asexually

• 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 28

What are 4 animal cloning methods?

Answer 28

1. Binary fission
2. Budding
3. Fragmentation
4. Parthenogenesis

Question 29

What is binary fission?

Answer 29

• 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 30

What is budding?

Answer 30

• 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 31

What is fragmentation?

Answer 31

• 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 32

What is parthenogenesis?

Answer 32

• 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

Question 33

What is vegetative propagation?

Answer 33

Small pieces of the plant can be induced to grow independently

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 34

What are some examples of types of roots and shoots that are capable of vegetative propagation?

Answer 34

• 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 35

How do some plants (mainly algae, mosses and ferns) reproduce?

Answer 35

Asexually by producing spores

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

Question 36

How do human beings capable of creating genetic clones through natural means?

Answer 36

• 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

Question 37

What cell will differentiate to form all the different tissues comprising the organism

Answer 37

Embryonic cells (pluripotent: (meaning they can divide and become any type of tissue)

Question 38

What happens if you separate embryonic cells artificially in a laboratory? What to do with it next? How about naturally?

Answer 38

• Each group of cells will form cloned organisms
• The separated groups of cells are then implanted into the uterus of a surrogate to develop into genetically identical clones

This separation of embryonic cells can also occur naturally to give rise to identical (monozygotic) twins

Question 39

How is the method of cloning using embryonic cells limited?

Answer 39

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

Question 40

What is a more reliable method of artificial cloning instead of via embryonic division? What does it involve?

Answer 40

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)

Question 41

What are the two main purposes of using differentiated cells to generate cloned embryos?

Answer 41

• 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 42

What is a stem cutting?

Answer 42

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

Question 43

Where are internodes? Where are stem cuttings placed?

Answer 43

• The region between nodes

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

Question 44

Why is stem cutting a common method used?

Answer 44

• Stem cutting is a common method employed to rapidly propagate plant species (including sugar cane, grapes and roses)

Question 45

What determines a successful rooting of a stem cutting? (7)

Answer 45

• 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)