Gel electrophoresis

Gel electrophoresis is used to separate DNA fragments. Electrophoresis uses an electric current to separate different-sized molecules in a sponge-like matrix. Smaller molecules move more easily through the gel pores than larger molecules.

Agarose gel can be used to separate DNA. First the gel is submersed in a tank filled with a salt solution which conducts electricity.

Using a pipette, DNA samples are loaded into slots made in the gel. The DNA is colorless but a blue dye is added, this makes it easier to track the DNA and its migration through the gel.

The phosphate groups in the DNA backbone carry negatively charges oxygen’s, giving DNA an overall negative charge. In an electric current, the negatively charged DNA moves towards the positive pole of the electrophoresis chamber.

DNA molecules then move through the gel by ‘reptation’-a reptile like snaking action through the pores of the gel. Smaller DNA fragments migrate faster and further over a given period of time than do larger fragments. This is how DNA fragments can be separated by size in the gel.

The introduction of a florescent dye, ethidium bromide, was used to stain the DNA. Ethidium bromide binds to the DNA double helix and glows in ultra-violet light. This lets researchers see where the separated DNA fragments end up.

DNA Profiling

Genes make up the blueprint for our bodies and almost every cell in the human body contains a copy of the blueprint, stored inside the nucleus. These genes are on the DNA on strands known as chromosomes. Certain portions of DNA are unique to each individual and DNA profiling is a way of establishing identity and is used in a variety of ways, such as finding out whether twins are fraternal or identical. DNA samples are usually obtained from blood.

DNA profiling is a technique used by forensic scientists to identify an individual by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier.

DNA profiling involves the (partial) sequencing of genomes. Profiles tend to focus on areas of satellite of junk DNA which vary significantly between individuals. Junk DNA is used because by sequencing a number of sections a unique ‘genetic fingerprint’ can be created for an individual.

The uses for profiling DNA:

•             Paternity - to find out if the alleged father is actually the biological father of the child.

•             Twins - identical twins share the same genetic material, while fraternal (non identical) twins develop from two eggs fertilised by two sperm and are no more alike than individual siblings born at different times. It can be difficult to tell at birth whether twins are identical or fraternal.

•             Siblings - for example, adopted people may want to have DNA tests to make sure that alleged biological siblings are actually their blood brothers or sisters.

•             Immigration - some visa applications may depend on proof of relatedness.

•             Criminal justice - DNA testing can help solve crimes by comparing the DNA profiles of suspects to offender samples. Victorian law allows the collection of blood and saliva samples from convicted criminals and suspects. DNA profiles are then kept on a database.

Advantages of DNA profiling:

•             DNA tests can be applied to any human sample that contains cells with nuclei, such as saliva, semen, urine and hair.

•             DNA tests are extremely sensitive, and can be conducted using samples that would be too small for other serological tests.

•             DNA resists degeneration even after contamination with chemicals or bacteria.

•             The ability of DNA profiling to exclude a suspect means the police are able to confidently drop that line of enquiry and continue their investigation down other avenues.

Limitations of DNA profiling

•             DNA profiling can give incorrect results, due cross-contamination of samples.

•             Old DNA profiling technologies are more prone to errors, which could give false-negative or false-positive results.

•             DNA profiles can only offer statistical probability (for example, one in a million), rather than absolute certainty.

•             The more people tested, the lower the statistical probability. For example, the probability of one in a million may nosedive to one in 10,000 if enough people are profiled for a single test.

•             DNA databases stored on computer are vulnerable to exploitation via hackers.

•             Some critics point out that holding a person’s DNA profile on record is, in a sense, a violation of that person’s DNA ‘ownership’.

•             DNA evidence is easily planted at a crime scene.

4.4.1 Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA.

PCR is the cloning or amplification of DNA, copies need to be made to rapidly increase the amount of DNA, this is useful if the source of DNA is small.

Firstly in a PCR reaction, DNA samples are heated to 94-96⁰C for several minutes to denature (separate into single strands) the target DNA.

The temperature is then lowered to 50-65⁰C for several minutes to let left and right primers to base pair to their complementary bases. The primers are designed to bracket the DNA region to be amplified.

Temperature is then raised again to 72⁰C for several minutes to allow Taq polymerase to attach at each priming site and synthesize a new DNA strand.

Temperature is raised to 94-96⁰C again to denature the DNA like in the beginning thus starting the process again

Animation: http://www.dnalc.org/resources/animations/pcr.html

Why might you want to amplify minute quantities of DNA?

We need to clone DNA for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. 

Past Paper Questions on Bio engineering

Discuss the ethical arguments for and against the cloning of humans [4]

The definition of clone is a group of genetically identical organisms derived from a single parent. These clones are made using laboratory techniques and starts with the production of human embryos. Therefore there are many issues with cloning; some arguments against it are that generating a new human embryo just for research is unnatural and wrong and is opposed by some religions. Cloning humans could also reduce the value of the human and clones may have health problems and they are more likely to die from complications. Cloning is also an expensive process and not fully successful so there could be better allocation of resources

On the other hand, the arguments for cloning are that it may help provide tissues and skin to repair burns, organs for transplantation and can lead to future medical breakthroughs.

Outline a basic technique for gene transfer involving plasmids [5]

Gene transfer involves plasmids which is a small piece of circular DNA. The plasmid is first removed from a cell and cut with restriction enzymes. The genes from another organism is also cut with the same restriction enzyme and spliced together with the plasmid using DNA Ligase. This plasmid is called a recombinant DNA which is then put into the genome of a host cell and then a fermenter to be cloned.

Outline a technique for transferring genes between species [5]

Gene transfer between species is when a gene from one organism is taken and placed in another organism. Firstly the gene for transfer has to be obtained; this is done by using restriction enzymes to cut out the useful gene. A plasmid, small circular DNA molecules found in bacteria, are cut with the same restriction enzyme and the gene cut out is spliced together with the plasmid using DNA Ligase. This plasmid is called a recombinant DNA which is then put into the genome of a host cell and then a fermenter to be cloned. The bacteria with the Recombinant DNA are then grown by asexual reproduction.

4.4.10 Discuss the potential benefits and possible harmful effects of one example of genetic modification.

A genetically modified organism (GMO) is an organism containing a transplanted gene.

An example is in the genetically modified salt tolerance in tomatoes. Plants like tomatoes couldn’t grow in salty conditions since this hypertonic soil water results the death of the plant however once the gene for salt tolerance was introduced in the tomato it now has the opportunity to be grown in saltier areas.

Benefits of GMO:

§  Increased yields particularly in regions of food shortage.

§  Yields of crops with specific dietary requirement such as vitamins and minerals.

§  Crops that won’t spoil so easily during storage.

§  Higher meat yields in genetically modified animals thus reducing hunger.

§  GM breeding is faster more specific and certain.

§  Plants are genetically modified to resist pests and extreme weather conditions which extends its survival.

Disadvantages and concerns of GMO:

§  Genetically modified animals and plants are considered un-natural and unsafe for human consumption.

§  There is a risk of the escape of 'genes' into the environment where they can be passed to other organisms with unknown effects. The genes could give an unnatural advantage for certain species over others.

§  There is a risk for allergies from the GM plants and animals.

§  It is worrisome that most of the food in the world could be controlled by a small number of corporations.

§  Decrease in biodiversity.

§  Gene transfers can be potentially harmful to animals

4.4.8 Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase.

Gene transfer is when a gene from one organism is taken and placed in another organism.

Stage 1 is obtaining the gene for transfer. This is done by using restriction enzymes to cut out the useful gene that is to be transferred; this leaves sticky ends on each side of the gene.

Stage 2 is preparing a vector for the transferred gene. This is done by using plasmids which are small circular DNA molecules found in bacteria. These are cut with the same restriction enzyme and this leaves the same complementary 'sticky ends' in the plasmid like the ones on the gene.

Stage 3 is the when gene has to be copied and this is when the gene is combined with the DNA. The gene is glued into the plasmid using DNA ligase. This plasmid is called a recombinant DNA and it can be used as a vector.

Stage 4 is the isolation of the transformed cells. The vector is then put into the genome of a host cell (bacterium, yeast or other cell) as the bacteria gives the plasmid the ideal conditions to grow and this is done by putting it into a bioreactor. Many of the cells remain untransformed but some of the cells are transformed to contain the recombinant DNA and these transformed cells will be separated from untransformed. 

Stage 5 is obtaining the final product. This is done when the transformed cells are isolated and put into a fermenter the right to be cloned. The bacteria with the Recombinant DNA are then grown by asexual reproduction and the final step is to isolate and purify the product called downstream processing.

E.6.7 Outline two examples illustrating the adaptive value of rhythmical behavior patterns.

North American flying squirrels

They are adapted to fly at night because at night there is the most food and the least competition. Its pattern is regulated by an internal biological clock and not by external clues because even though the hours of darkness change throughout the year in North America, the flying squirrel continues to fly out at the same time no matter if it is light or dark. But external clues such as the light do come in helping the ground squirrel regulate its biological clock and adapt to its changing environment.

Fiddler Crabs

This crab only mates during a full moon or new moon as it is related to the best tidal periods.  When the moon is full or new, the gravitational pull of the moon and sun are combined. At these times, the high tides are very high and the low tides are very low. These are called Spring tides. The adaptive value of this behavior is that the mating is timed to coincide with a Spring tide so the eggs can be released during a nocturnal high spring tide so that the tiny, swimming larvae are washed far out to sea. They develop out there for several weeks and then wash into shore. 

The Human Brain

Drugs

Excitatory drugs

Nicotine
• Nicotine in tobacco products is a stimulant which mimics acetylcholine (Ach). Thus, it acts on the cholinergic synapses of the body and the brain to cause a calming effect. After Ach is received by the receptors, it is broken down by acetyl cholinesterase but the enzyme cannot break down the nicotine molecules which bind to the same receptors. This excites the postsynaptic neuron and it begins to fire, releasing a molecule called dopamine. Dopamine gives the feeling of pleasure, a molecule of the ‘reward pathway’ of our brains.

Amphetamine
• Stimulates transmission at adrenergic synapses and gives increased energy and alertness. Amphetamine acts by passing directly into the nerve cells which carry dopamine and noradrenalin
• It moves directly into the vesicles of the pre synaptic neuron and causes their release into the synaptic cleft. Normally, these neurotransmitters would be broken down by enzymes in the synapse, but amphetamines interfere with the breakdown.
• Thus in the synapse high concentrations of dopamine cause euphoria, and high concentrations of noradrenalin may be responsible for alertness and high energy effect of amphetamines.

Inhibitory Drugs 

Benzodiazepine
• Reduces anxiety can also be used against epileptic seizures.
• Its effect is to modulate the activity of GABA which is the main inhibitory neurotransmitter. When GABA binds to the postsynaptic membrane, it causes Chloride ions to enter the neuron. 
• This hyperpolarizes the neuron, and resists firing.
• Benzodiazepine increases the binding of GABA to the receptor and causes the post synaptic neuron to become more hyperpolarized.

Alcohol
• Inhibitory neurotransmitters, called GABA, are active throughout the brain. These neurotransmitters act to control neural activity along many brain pathways. When GABA binds to its receptors, the cell is less likely to fire.
• However, in another area of the brain, another neurotransmitter called glutamate acts as the brain’s general-purpose excitatory neurotransmitter.
• When alcohol enters the brain it delivers a double sedative punch. First, it interacts with GABA receptors to make them even inhibitorier.
• Second, it binds to glutamate receptors, preventing the glutamate from exciting the cell. 
• Alcohol particularly affects areas of the brain involved in memory formation, decision-making and impulse control.

Cocaine (Excitatory Drug):

Cocaine is a psycho-active drug along with THC (Tetrahydrocannabinol) they affect a mood of a person as they both concentrate on the reward pathways in the brain. In the reward pathways a pleasuable mood enhancing sensation is produces. The cause to this natural 'high' is the secretion of the neurotransmitter dopamine. Dopamine receptors are found in the post synaptic membrane which when activated depolarizes the post synaptic neurone in regions of the brain associated with a feeling of pleasure. Since dopamine is the neurotransmitter in the ‘reward pathway’, the longer it stays in the synapse the better you feel. 

Cocaine blocks Dopamine transporters by attaching to the presynaptic dopamine pumps, leaving dopamine trapped in the synaptic cleft, thus dopamine binds again and again to receptors which over stimulates the cell. Thus cocaine results in post synaptic excitement of cholinergic synapses which are associated will elevated levels of activity. Cocaine is described as creating a mood of euphoria and cocaine users are often described as fidgety and energetic. This drug can also increase body temperature, blood pressure and heart rate. Users risk heart attacks, respiratory failure, strokes, seizures, abdominal pain and nausea. 

Marijuana or Tetrahydrocannabinol (THC) (Inhibitory Drug):

THC is the main psychoactive chemical in marijuana.
Before marijuana enters the system, inhibitory neurotransmitters are active in the synapse. These neurotransmitters inhibit dopamine from being released. When activated by the body’s own native cannabinoid (called anandamide), cannabinoid receptors turn off the release of inhibitory transmitters. Without inhibition, dopamine can be released. THC, the active chemical in marijuana, mimics anandamide and binds to cannabinoid receptors. Inhibition is turned off and dopamine is allowed to squirt into the synapse.

Anandamide is known to be involved in removing unnecessary short term memories. It is also involved for slowing down movement, making us feel mellow and calm. Unlike THC, anandamide breaks down very quickly in the body. That explains why anandamide doesn’t produce a perpetual natural ‘high’.

Explain how pre-synaptic neurons can affect post-synaptic transmissions of impulses [7]

Firstly a nerve impulse coming into the pre synaptic neuron would cause calcium ions to diffuse through channels in the membrane. The calcium influx then causes vesicles containing neurotransmitters to fuse with the pre synaptic membrane, this process is called exocytosis. The neurotransmitters are then released into the synaptic cleft and diffuse across it to bind with the post synaptic neuron. This is called an excitatory synapse. The binding then cause’s ion channels to open and sodium ions will then diffuse through this channel down the concentration gradient. This then initiates the action potential which begins to move down the post-synaptic neuron because it has been depolarized. 

Opposite from an excitatory synapse is an inhibitory synapse. This is when the release of neurotransmitters into the cleft inhibits an action potential being generated in the post-synaptic neuron. This means that instead of the neurotransmitters triggering the opening of ion channels to let sodium out it will instead allow chlorine ions to enter the neurone or potassium to leave. This will then make the post-synaptic neurone more negative (hyperpolarised) and therefore less likely to initiate an action potential.


Mark scheme

a)      Pre synaptic neuron can be excitory of inhibitory

b)      Chlorinergenic neurons release acetylcholine

c)       Found in neuromuscular junctions/in autonomic nervous system/most junctions in voluntary nervous system

d)      Adrenic nerons release noradrenaline

e)      Found in sympathetic pathways (in brain)

f)       Both types of neurons can be excitory

g)      Neurotransmitters (NT) bind to receptors on post synaptic membrane

h)      Triggers opening of Na+ gates/channels/Na+ moves across membrane

i)        Causes depolarization

j)        NT’s are degraded/destroyed or recycled e.g. acetyl choline esterase breaks down acetyl choline

k)      Other inhibitory NT’s e.g. GABA (cocaine, alcohol), Dopamine

l)        Inhibitory NT’s less permeable to Na+/cause Cl- ions to diffuse in

m)    Hyper-polarization

n)      By K+ diffusing out 

Explain the process of synaptic transmission [7]

A nerve impulse would first travel to end of pre synaptic neuron, this will then trigger a influx of calcium ions. In the pre synaptic neuron there are swollen membranous areas called terminal buttons and within these there are many vesicles filled with neurotransmitters. The calcium influx then causes synaptic vesicles containing neurotransmitters to fuse with the pre synaptic membrane by exocytosis. The neurotransmitters are then released into the synaptic cleft and diffuse across it to bind with the receptors on the post synaptic neuron. This then causes ion channels to open on neuron and sodium diffuses into postsynaptic neuron. This initiates the action potential to begin moving down the postsynaptic neuron because it’s been depolarized. The neurotransmitter is then degraded and broken into two or more fragments by specific enzymes. They’re then released from the receptor protein. The ion channel closes to sodium ions. The neurotransmitter fragments diffuse back across the synaptic gap to be reassembled into the terminal buttons of the pre synaptic neuron. 


Mark scheme

a)      Pre synaptic neurons pass a stimulus to post-synaptic

b)      Pre synaptic releases NT into cleft

c)       Process involves exocytosis

d)      Exocytosis triggered by Ca2+ into neuron (bulb)

e)      NT binds with receptor on postsynaptic cleft

f)       NT binding causes ion channels to open

g)      Ions diffuse into/out of cell

h)      Depolarization or Hyper-polarization

i)        Outcome depends on type of receptor

j)        E.g. Na+ going into postsynaptic neuron = depolarization

k)      Cl- passing into the post synaptic neuron = hyper polarization

l)        NT destroyed/deactivated by enzymes

Plants are classified together in a kingdom. Other organisms are classified in other kingdoms. Outline the value of classifying organisms [4]

a) Shows how organisms are related

b) Helps cope with the large number of organism

c) Easier to store/find information

d) Easier to find useful organisms (e.g. drugs in animals)

e) Makes it easier to ID organisms

f) Allows predictions to be made about the nature of a organism

g) Traces possible evolutionary links

h) ID homologous structures

Outline the consequences of rising carbon dioxide concentrations in the Earth’s atmosphere [5]

The rising carbon dioxide concentrations in the atmosphere create a blanket over the Earth which keeps the suns wavelengths in the atmosphere; this would then increase global warming as the Earth would continually heat up as it cannot escape due to the build up of gases. With increased heat come many consequences such as the melting of polar ice caps, this will then in turn cause a rise in global sea levels causing flooding in low lying areas. With the heat there can also come natural disasters such as drought which would affect agriculture leading to a decline in food production which has the consequence of decreasing life expectancy. The heat also brings about diseases from mosquitoes, such as malaria and dengue fever, because they thrive in heat. Also warmer temperatures allow pathogens to survive better.

Explain the light-independent reaction of photosynthesis [8]

The energy in the form of ATP and NADPH made in the light dependent reaction is then used in the light independent reaction to fix carbon from carbon dioxide into organic molecules. This reaction is called the Calvin cycle and it takes place in the stroma of the chloroplast and is controlled by enzymes. Firstly a single carbon in carbon dioxide is fixed with RuBP and is catalyzed by the enzyme Rubisco to form 2 molecules of Glycerate-3-phosphate. The Glycerate-3-phosphate is then reduced to triose phosphate by using the energy which comes from the oxidation of NADPH and ATP. Triose phosphate is then used to regenerate Ribulose Bisphosphate. 

The carbon cycle involves both the production and the fixation of carbon dioxide. Draw a labelled diagram to show the processes involved in the carbon cycle [5]

Explain the process of aerobic respiration including oxidative phosphorylation [8]

In aerobic respiration, glucose is split into pyruvate by glycolysis. The pyruvate then enters the mitochondria and reacts with coenzyme A by the process of oxidative decarboxylation to form acetyl CoA, in this process carbon dioxide and NADH is formed. The acetyl group then enters the kreb cycle where FAD+ and NAD+ accept hydrogen to form NADH and FADH2. The energy in these molecules is then used to make many more molecules of ATP. In the inner membrane of the mitochondria there are the proteins. Both NADH and FADH2 donate electrons to the electron transport chain. As electrons move from 1 complex to another they transfer their energy to pump protons across the membrane, this then creates a concentration gradient across the membrane. Oxygen is the final electron acceptor; it will then combine with the protons to form water. 

Describe the process of active transport [4]

Active transport is when a protein moves a certain material across the membrane from a region of lower concentration to a region of higher concentration. This means the substance is absorbed against the concentration gradient and energy is needed for this active transport to work. The energy is usually comes from adenosine triphosphate or ATP, every cell supplies its own ATP by cell respiration. Globular proteins or pump proteins or transporter proteins in membranes carry out the active transport, the membrane must contain a lot of these proteins so that the cell can control the contents of its cytoplasm precisely. An example of active transport is in human nerve cells where potassium ions are pumped in and sodium ions are constantly transported out of the cell by active transport into the external fluid bathing the cell to build up a store of potential energy or an electrical impulse that is used to transmit a nerve impulse.

Draw a diagram of the ultrastructure of an animal cell as seen in a electron micro graph [6]

E.2.2 Label a diagram of the structure of the human eye.

E.2.6 Label a diagram of the ear and E.2.7 Explain how sound is perceived by the ear

E.2.4 Compare rod and cone cells.

Photoreceptors are the rods and cones

Both rods and cones synapse with their bipolar neurons

Rod cells are sensitive to light. They receive the stimulus of light, even dim light, and synapse with a bipolar neuron

Cone cells are activated by bright light. They receive the stimulus of bright light and synapse with a bipolar neuron

Bipolar neurons: cells in retina which carry impulses from a rod or cone cell to a ganglion cell of the optic nerve. They are called bipolar because they each have 2 processes extending from the cell body.

Ganglion cells are the cell bodies of the optic nerve. They synapse with the bipolar neurons and send the impulses to the brain.

Rods	Cones
Low/Dim light	Bright light
1 type of rods found in retina. It can absorb all wavelengths of visible light	3 types of cones found in retina. 1 can absorb red light, 1 blue and 1 green
Impulses from a group of rod cells pass a single nerve fibre (Ganglion axon) in the optic nerve	Impulse from a single cone cell passes to a single nerve fibre in the optic nerve

E.2.1 Outline the diversity of stimuli that can be detected by human sensory receptors.

Stimuli are detected by receptors which then trigger sensory neurones to transmit info to the CNS

Different receptors detect different stimuli

Human receptors can be classified as mechanoreceptors, thermo receptors, chemoreceptor’s or photoreceptors

Form of energy detected	Type of receptor	Location in body
Thermoreceptors (heat energy)
Temperature change in the skin	Cutaneous receptors	Dermis of skin
Temperature change in the body	Cells of hypothalamus	Brain
Chemoreceptors (chemical stimulation)
Detects chemicals in the air (smell)	Epithelium of the nasal cavity	Nose
Detects chemicals on the tongue (taste)	Taste buds	Tongue
Detects changes in the composition of arterial blood	Carotid body	Located near the fork of the carotid artery
Osmotic concentration of blood	Osmoreceptor	Kidneys
Mechanoreceptors (movement)
Light Touch	Touch Receptors	Mostly in skin dermis
Touch Pressure	Pressure receptor	Skin
Changes in length of muscle	Stretch receptors such as muscle spindles	Muscle
Sound and balance	Sensory hair cells	Auditory system (ears)
Pressure of blood flowing through vessels	Baroreceptors	Blood vessels
Photoreceptors (light)
Light	Rods and cones	Eye