Biotechnology and gene technology Flashcards
1
Q
What is the basis of asexual reproduction in eukaryotes?
What about in prokaryotes?
How do genetic differences arise in organisms that reproduce asexually?
A
Mitosis
Binary fission
Via mutations
2
Q
Define vegetative propagation
A
The production of structures in an organism that can grow into new individual organisms, they are genetically identical to their parents and are therefore clones
3
Q
Define clone
A
Genes, cells or whole organisms that carry genetically identical material as they are derived from the same original DNA
4
Q
How are identical twins produced?
A
They are produced when a zygote splits in 2, they are natural clones
5
Q
Finish the sentence…
When organisms reproduce asexually all of the offspring are…
A
…genetically identical and are clones with cloned DNA
6
Q
Can cloning occur naturally? Explain
A
Yes because it’s essential for growth and reproduction
7
Q
There is a distinction between cloned genes, cloned cells and cloned organisms. Give examples of when each of the occur.
A
Cloned genes: DNA replication, protein synthesis
Cloned cells: Mitosis for growth of any multicellular organism
Cloned organisms: Identical twins, offspring from asexual reproduction
8
Q
Briefly describe binary fission. Are the offspring genetically identical or not?
A
It occurs in prokaryotes, DNA replicates and then the cell divides into 2.
The offspring are genetically identical to each other and their parents
9
Q
Briefly describe mitosis
A
When DNA replicates then separates to form 2 new nuclei and then 2 genetically identical daughter cells are formed
10
Q
In multicellular organisms, especially plants, what can happen to the cells produced via mitosis?
A
They can grow into new separate organisms which are genetically identical to the parent and they are therefore clones. This is vegetative propagation.
11
Q
Give three advantages of asexual reproduction
A
- It’s quick so organisms can reproduce rapidly, allowing them to take advantage of their environment
- It can be completed if sexual reproduction fails or isn’t possible
- All of the offspring have the genetic information in order to survive in their environment
12
Q
Give 1 disadvantage of asexual reproduction
A
There is no genetic variation so all the offspring will have the genetic parental weaknesses. This means that if the environment changes, all of the organisms will be equally susceptible to death.
13
Q
What happens when English Elm is damaged due to disease or burning?
A
Vegetative propagation: It reproduces asexually allowing the species to survive catastrophes. Root suckers/basal sprouts appear within 2 months of the destruction, they grow from the meristem tissue in the trunk close to the ground because this is where the least damaged is likely to have occurred.
14
Q
Give 1 detailed advantage of vegetative propagation in English Elm trees
A
The root suckers can help the elm spread because they form a clonal patch (a circle of root suckers) after the Elm dies due to stress or felling. These clones will then produce more root suckers and the Elm clones will spread.
15
Q
Give 1 detailed disadvantage of vegetative propagation in English Elm trees.
A
Dutch Elm disease, which has spread through Europe, kills the leaves of an Elm, then the branches, then the trunks. The fungal disease is spread via a beetle. When an English Elm gets infected, it grows root suckers but because there is no genetic variation, the root suckers get infected also. This means that natural selection doesn’t occur.
16
Q
Define tissue culture
What type of tissue can be grown?
What type of tissue is usually grown?
Why?
A
The separation of cells from a tissue and their growth in or on a nutrient medium.
Any type
Undifferentiated callus tissue because it can, with the addition of hormones, stimulate the development of a complete plant.
17
Q
Describe the 2 main methods of artificial vegetative propagation
A
- Taking cuttings: A section of stem is cut between nodes, the cut end is then treated with root growth promoting hormones. You then plant this cutting and it grows into an individual new organism. Large numbers of plants can be produced quickly from this method
- Grafting: A shoot section from a woody plant is joined to an already growing rootstock. This grows an individual organism. The graft is genetically identical to the parent but not to the rootstock. Side grafting is when you cut a slit in the plant and slide the graft into the slit.
18
Q
Describe 2 disadvantages of grafting and cuttings
A
They don’t always reproduce well
They can’t produce huge numbers of cloned plants
19
Q
Name a modern method of artificial vegetative propagation and list 3 advantages of it.
A
Tissue culture
It can produce huge numbers of genetically identical plants from a very small amount of plant material, it can generate large stocks very quickly and the plants are known to be disease free
20
Q
What is the most common method for large scale cloning in plants?
What plant is usually cloned?
A
Micropropagation
Orcids
21
Q
Describe the 6 stages of micropropagation.
A
- An explant (the shoot tip) is taken from a plant to be cloned
- The explant is placed in a nutrient medium
- The cells divide but don’t differentiate, which means that they form a callus (a mass of undifferentiated cells)
- After a few weeks, single callus cells can be removed from the mass and placed on a growing medium containing hormones that promote shoot growth
- After a few more weeks, the growing shoots can be placed onto a different growing medium with different concentrations of hormones that promote root growth.
- The plants are then transferred to a greenhouse where they then acclimatise before being planted outside.
22
Q
Is micropropagation a form of genetic engineering?
A
No because genes aren’t added or removed
23
Q
Define cloned animal
A
One that has been produced using the same genetic information as another animal. It has the same genotype as the donor.
24
Q
Define totipotent stem cells
A
Cells that are naturally capable of going through the stages of development to generate a new individual. In animals, only embryonic cells are capable of this. They’re able to switch on any of the genes present in the genome.
25
List the two methods of artificial cloning in terms of animals
Splitting embryos and nuclear transfer.
26
Describe the procedure for splitting an embryo
An egg and sperm goes through in vitro fertilisation, and the embryo is grown in vitro also. When the embryo gets to 16 cells, the cells from the developing embryo can be separated out and implanted in a surrogate mother. This produces separate genetically identical organisms. It has been used on sheep, cattle, rabbits and toads
27
Describe in detail the procedure for nuclear transfer
A differentiated cell from an adult can be taken, and its nucleus can be placed in an enucleated egg cell via electro-fusion. The egg is then cultured in an oviduct. After this, it's implanted into a surrogate mother and it then goes through the stages of development using the genetic information from the inserted nucleus. This method has been used on sheep.
28
List 3 advantages of cloning animals
1. High value animals (high yields) can be cloned in large numbers
2. Rare animals can be cloned to preserve the species
3. Advantageous genetically modified animals (sheep that produce pharmaceutical chemicals) can be quickly reproduced
29
List 3 disadvantages of cloning animals
1. High value animals aren't always produced with animal welfare in mind, e.g. meat producing chickens that are unable to walk.
2. Genetic uniformity reduces variation so plants wouldn't be able to cope with or adapt to changes in the environment
3. Cloning animals via nuclear material might affect the health of the animals' cells long term. E.g. the first cloned sheep had premature ageing and had to be put down.
30
What can cloned cells be used for?
They can be used to generate cells, tissues and organs to replace those damaged by diseases or accidents
31
List 4 advantages of cloning cells
1. They will be genetically identical to the individuals own cells and won't be rejected by the immune system
2. It could end problems like waiting for donor organs
3. They are totipotent so they can be used to generate any type of cell which is good because currently some damage can't repaired via transplantation.
4. It's less dangerous than major operations/transplantations
32
Define non reproductive cloning
Cloning that's used to produce some parts of the body rather than creating a whole organism which is what reproductive cloning does.
33
Give 3 examples of what non-reproductive cloning can be used for
1. The regeneration of heart muscles after a heart attack
2. The repair of nervous tissue that's destroyed by multiple sclerosis
3. The repairing of the spinal cord that's been paralysed due to an accident
34
What is another term for non-reproductive cloning?
| Give 2 disadvantages for non-reproductive cloning
Therapeutic cloning
Ethical issues when using embryonic material
Lack of understanding about how cloned cells will behave over time.
35
Define biotechnology
It says it in the name. It exploits living organisms/biological processes to improve agriculture, food science, animal husbandry, medicine and industry.
36
List 4 reasons for using biotechnological processes for commercial uses.
To produce foods
To produce drugs/pharmaceutical chemicals
To produce enzymes/commercial chemicals
For the bioremediation of waste products.
37
Give 3 examples of foods that are produced via biotechnological processes
Describe the process and the organisms involved
1. Cheese and yoghurt - Lactobacillus is grown in milk which changes the texture and flavour of milk. It also prevents other bacteria from growing, which preserves the food
2. Mycoprotein/Quorn - Fusarium fungus is grown in a culture which produces mycelium fungus. This is then separated and processed as food.
3. Soya sauce - Roasted soya beans are fermented with fungus or yeast, e.g. Aspergillus
38
Give 2 examples of drugs that are produced via biotechnological processes
Describe the process and the organisms involved
1. Penicillin, an antibiotic - Penicillium fungus is grown in a culture and it produces penicillin as a by-product of its metabolism
2. Insulin - E. coli bacteria are genetically modified to carry the human insulin gene and the bacteria produces insulin as it grows.
39
Give 3 examples of enzymes/commercial chemicals that are produced via biotechnological processes
Describe the process and the organisms involved
1. Pectinase, used in fruit juice extraction - A. niger fungus is grown in certain conditions to produce and secrete pectinase
2. Calcium citrate, for detergents - A. niger fungus produces citric acid as a by-product of its normal metabolism
3. Bio-gas fuel production - Methanogenic bacteria is grown on concentrated sewage. It respires anaerobically and generates gases that can be used for fuel.
40
Give 1 example of the bioremediation of waste products that uses a biotechnological process.
Describe the process and the organism involved
1. Waste water treatment - A variety of bacteria/fungi use the organic waste in water as a source of nutrients, this makes the waste harmless. For example, Fusarium is a waste product of the corn milling industry
41
What type of organism is often used for biotechnological processes?
Microorganisms like bacteria and fungus
42
Give 5 out of 7 reasons as to why microorganisms are most commonly used in biotechnological processes
1. They grow rapidly in favourable conditions with generation times as little as 30 minutes
2. They often produce proteins and chemicals that can be harvested
3. They can be genetically engineered to produce specific products
4. They grow well at relatively low temperatures, much lower than those needed for chemical engineering
5. They aren't dependent on climate so can be grown anywhere in the world
6. They generate more pure products in comparison to chemical engineering
7. They can be grown using materials that are useless or toxic to humans.
43
Define culture
A growth of microorganisms. It can be 1 species aka pure culture or it can be a variety aka mixed culture. It can be grown in a nutrient broth or on a nutrient agar gel.
44
Define closed culture
The growth of microorganisms in an environment where all conditions are fixed and contained. No new materials are added and no waste products/organisms are removed
45
What is the standard growth curve?
The series of events that occur in a closed culture of microorganisms. This can be plotted on a graph
46
What are the 4 phases of a standard growth curve in terms of a closed culture?
Describe each phase.
Lag phase: Organisms are adjusting to their environment, they are taking in water, expanding cells, activating genes, synthesising enzymes etc. They are active but not reproducing so the population remains fairly constant.
Log phase: Also called the exponential phase. The population size doubles each generation as there's enough space and nutrients to reproduce. The population (in some bacteria) can double every 20-30 minutes. Just after this phase, there are less resources so secondary metabolites are produced, these are antibiotics (e.g. penicillin) and they kill off surrounding bacteria to reduce competition.
Stationary phase: Nutrient levels decrease and waste products like CO2/other metabolites increase. Organisms die at the same rate that they are being produced. In an open culture, this would be the carrying capacity.
Death/decline phase: Nutrient exhaustion and increased levels of toxic waste products/metabolites lead to the death rate being higher than the production rate. Eventually all organisms die.
47
Define the 2 definitions for fermentation
1. The use of anaerobic respiration to produce substances, e.g. ethanol from yeast which is a natural by-product of anaerobic respiration.
2. The culturing of microorganisms in aerobic or anaerobic fermentation tanks. The useful substances that are generated are separated and treated.
48
List 3 things that are produced from metabolic processes
1. New cells/cellular components
2. Chemicals like hormones and enzymes
3. Waste products which vary depending on the organism
49
Define primary metabolites and describe how its production would be represented on a graph
Substances produced by an organism as part of its normal growth. E.g. Enzymes, amino acids, proteins, ethanol, lactate and nucleic acids. On a graph, the production of primary metabolites should match the growth of a population as every organism needs these molecules to survive.
50
Define secondary metabolites and describe how its production would be represented on a graph
Substances produced by an organism that aren't part of its normal growth, e.g. penicillium producing the secondary metabolite penicillin. On a graph, it doesn't match the growth rate because they are only produced after the main growth period.
51
Do a lot of or not many microorganisms produce secondary metabolites?
Not many microorganisms produce secondary metabolites but all of the produce primary ones.
52
Define an aseptic technique
Any measure taken at any point in a biotechnological process to ensure that unwanted microogranisms don't contaminate the culture/products.
53
Define asepsis
The absence of unwanted microorganisms
54
What is usually used in order to produce the growth of a particular microorganism on an enormous scale
A industrial-scale fermenter.
55
What is an industrial scale fermenter?
A huge tank that has a capacity of tens of thousands of litres. The growing conditions in it can be manipulated and controlled to ensure that the best possible yield is obtained.
56
What are the specific growing conditions of a microorganism?
It depends on the microorganism and whether the process is designed to produce primary or secondary metabolites.
57
What conditions need to be controlled when growing microorganisms? 4 things
1. Temperature - too hot the enzymes will be denatured, too cold the growth will be slowed
2. Type/time of addition of nutrient - A nutrient supply is required (e.g. carbon, nitrogen etc.) but the timing depends on whether you want primary or secondary metabolites.
3. Oxygen concentration - Most commercial applications grow organisms in aerobic conditions. A lack of oxygen will produce unwanted products from anaerobic respiration and also growth rates will be slowed
4. pH - Changes in pH can reduce the activity of enzymes which will reduce the growth rates.
58
How are starter populations obtained? These are needed for large cultures
By taking a pure culture and growing it in a sterile nutrient broth
59
List eleven features of a large scale industrial fermenter
1. Pressure vent- prevents gas build up
2. Sterile air inlet- provides oxygen for aerobic respiration
3. Mixing blades- to stir the culture
4. Water jacket inlet- allows a circulation of water around the tank to regulate temperature
5. Outlet tap- to drain the culture
6. Motor- to rotate the mixing blades
7. Inlet- for the addition of nutrients
8. Water jacket outlet- to remove water
9. Electronic probes- to measure oxygen, pH and temperature
10. Air outlets- release air into the culture, which mixes it, this is called sparging
11. Filters in all the inlets and outlets- this prevents contamination and ensures everything is sterile
60
What are the two types of cultures/ fermentation operations?
Bath cultures and continuous cultures
61
Describe a batch culture
The microorganism starter population is mixed with a specific quantity of nutrient solution and is then allowed to grow for a fixed period of time. No additional nutrients are added and this produces secondary metabolites. The fermentation tank is emptied after a set amount of time in order to get the secondary metabolites, which is usually penicillin.
62
Describe a continuous culture
When nutrients are added to the fermentation tank and products are removed at regular intervals. This process produces primary metabolites like insulin from genetically modified E. Coli
63
What are the advantages and disadvantages of a batch culture?
Advantages: easy to set up and maintain, if contamination occurs only one batch is lost, useful for producing secondary metabolites.
Disadvantages: growth rate is slower than in continuous cultures as nutrient levels decline, less efficient as it isn't in operation all if the time.
64
What are the advantages and disadvantages of a continuous culture?
Advantages: growth rate is higher than batch culture as there's a constant supply of nutrients, more efficient because the fermenter is always operating, useful for producing primary metabolites.
Disadvantages: set up is difficult and maintenance is hard as optimum growing conditions are constantly required, if contamination occurs huge volumes of product are lost.
65
What is a contaminant in terms of fermentations?
The growth of unwanted microorganisms in a culture
66
Give 5 reasons as to why a contaminant is bad in a culture?
1. They compete for space and nutrients with the culture microorganisms
2. They reduce the yield of useful products from the culture microorganisms
3. They can cause spoilage of the product
4. They can produce toxic chemicals
5. They could destroy the culture microorganisms and their products
67
List 3 aseptic techniques when growing a starter population
1. All apparatus are sterilised before and after use via steam sterilisation, UV light or heat from a flame.
2. Work is carried out in a fume cupboard/laminar flow cabinet to provide air circulation which carries the contaminants away from the work space
3. The culture is kept closed and away from the mech space whenever possible
68
List 4 aseptic techniques when using a large scale culture
1. Washing, disinfecting and steam cleaning the fermenter and pipes when not in use
2. The fermenter surfaces are made from polished stainless steal to prevent microbes sticking to surfaces
3. All nutrient media is sterilised before being added to the culture
4. Filters on all inlet and outlet pipes to avoid microorganisms entering the fermentation tank
69
Define immobilisation
Any technique where enzyme molecules are held, separated from the reaction mixture. The substrate molecules can still bind to the enzyme and the products formed go back into the reaction mixture
70
Name and describe two features that enzymes have which makes them useful in industrial processes
1. Specificity- they catalyse reactions between specific chemicals even when other chemicals are present. This means that there are fewer by-products and therefore less purification of the products is needed
2. Temperature of enzyme action- they function at relatively low temperatures in comparison to many industrial chemical processes which saves money on fuel costs.
71
Give an example of an organism that has enzymes which need a high temperature to function. These enzymes are sometimes used in industrial processes.
Thermophilic bacteria which thrive at high temperatures
72
How many products are usually required when carrying out clinical research/diagnosis/industrial processes?
Only one product is required from a single chemical reaction
73
Why is it more efficient to use isolated enzymes instead of growing whole organisms or using inorganic catalysts during clinical processes?
Because it would be a waste of time, energy and money to grow a whole organism for just one chemical reaction to produce one product.
74
Name a process that enables you to isolated an enzyme from a fermentation mixture. Can isolated enzymes be produced in large quantities from this process?
Downstream processing
| Yes
75
What is downstream processing?
A process that involves the separation and purification of any product from large scale fermentations
76
How can you easily achieve making enzyme-substrate complexes?
By mixing quantities of substrate and isolated enzymes together under suitable conditions
77
The product generated from enzyme substrate complexes needs to be extracted, is this a cheap or expensive process?
Expensive
78
Because extracting products from a mixture is expensive, what can you do instead which is cheaper?
You can immobilise enzymes so that they still catalyse enzyme-controlled reactions but they don't mix freely with the substrate and in turn the product. This means that extraction is cheaper.
79
When do enzymes and catalysts mix freely with each other?
| When don't they?
In cells or isolated systems
| When the enzymes have been immobilised
80
List 3 advantages of using immobilised enzymes
1. Enzymes don't mix with the products so purification/downstream processing costs are low.
2. Enzymes are immediately available for reuse which is useful in continuous processes
3. Immobilised enzymes are more stable because the immobilising matrix protects the enzymes
81
List 3 disadvantages of using immobilised enzymes
1. Immobilisation requires additional time, equipment and materials making it more expensive
2. Immobilised enzymes can be less active as they aren't mixing freely with the substrate
3. Contamination is costly to deal with as the whole process needs to be stopped
82
List 4 methods of immobilising enzymes
Adsorption, covalent bonding, entrapment and membrane separation
83
How do you know which method of immobilising enzymes to use?
It depends on the ease of preparation, cost, relative importance of enzyme leakage/discharge and efficiency
84
Describe the adsorption method when immobilising enzymes
The enzyme molecules are mixed with the immobilising support and they bind to the support via hydrophobic reactions and ionic links. The bonds are weak and enzymes can become detached (leakage). Nevertheless, adsorption can still give very high reaction rates. Adsorbing agents/support: porous carbon, glass beads, clays and resins
85
Describe the method of covalent bonding when immobilising enzymes
The enzymes covalently bond to a support and they covalently link with each other and with the insoluble molecule (support). They covalently link using a cross-linking agent like gluteraldehyde or sepharose. This method doesn't immobilise a large quantity of enzymes but the binding is strong so there is very little leakage. Insoluble material: clay particles
86
Describe the method of entrapment when immobilising enzymes
Enzymes are trapped in a gel bead or a network of cellulose fibres. They are trapped in their natural state as they don't bind to any other molecules which could affect their active site (adsorption and covalent bonding). Reaction rates are reduced because the substrates need to get through the trapping barrier which makes the active site less easily available compared to absorption and covalent bonding.
87
Describe the method of membrane separation when immobilising enzymes
Enzymes are physically separated from the substrate mixture via a partially permeable membrane. The enzymes are on one side and the substrate solution is passed along the other. The substrates are small enough to pass through the membrane and so are the products.
88
Define genomics
The study of the whole set of genetic information in the form of DNA base sequences that occur in the cells of organisms of particular species. Sequenced genomes and placed on public access databases
89
What does it mean if a gene is found in all organisms?
It means that it probably codes a protein that's essential for a fundamental life process
90
Give 4 examples of advances in science that use our understanding of the structure and role of DNA in organisms. Also, say when the method is used.
1. DNA profiling/genetic fingerprinting- it's used in forensic crime scene analysis and in paternity/maternity testing
2. Genomic sequencing/comparative genome mapping- it's used in research into the function of genes and regulatory gene sequences
3. Genetic engineering- it's used in the production of pharmaceutical chemicals, genetically modified organisms and xenotransplantation
4. Gene therapy- it's used to treat conditions like cystic fibrosis
91
Define xenotransplantation
Transplanting organs/tissues between members of different species
92
Is gene technology advancing rapidly or not much at all?
| Do gene technology techniques use natural or artificial processes?
It's advancing rapidly
| It uses natural processes
93
Give 5 examples of natural processes that are used for gene technology techniques
1. DNA strands being cut up into smaller fragments via restriction endonuclease enzymes
2. Fragments being separated into size via electrophoresis and then replicated many times using a polymerase chain reaction
3. DNA fragments being analysed to reveal their specific base sequence
4. DNA fragments being sealed back together via ligament enzymes
5. DNA probes locating specific sequences on DNA fragments
94
What is the point of gene technology?
To identify and manipulate sections of DNA including whole genes
95
What does coding DNA consist of?
What percentage of a genome is coding DNA?
What does the rest of the DNA consist of?
What is its function?
It consists if genes that code for polypeptides an proteins
1.5%
Non-coding DNA
It has many regulatory functions, for example producing RNA molecules
96
What is the purpose of genomics?
To study genomes and then compare the genes and regulatory sequences of different organisms in order to find out the role of genetic information in terms of health, behaviour and evolutionary relationships.
97
How many base pairs can be involved in a sequencing reaction? If there's more, the reaction can't operate
750 base pairs
98
Why does a genome need to be broken up in order to be sequenced?
Because a sequencing reaction can only occur when there is a maximum of 750 base pairs. A genome has many more than this.
99
Why is sequencing carried out a number of times on overlapping fragments?
To ensure that the assembled code is accurate which allows you to put the segments back together and form the completed code.
100
What are the three steps involved when preparing to sequence a genome?
1. Chromosomes are mapped to identify which part of the genome they have come from. This is done via looking at microsatellites which are short runs of repetitive sequences found in several thousand locations in a genome
2. Samples of the genome are sheared into smaller sections of 100000 base pairs. This is the shotgun approach
3. The sections are then placed into separate bacterial artificial chromosomes (BACs) and are transferred into E. coli cells. They're then grown in a culture to produce many copies. These cells are now clone libraries
101
Once the sections of genomes have been placed into BACs, what are the four stages of sequencing?
1. A number of specific BACs are firstly cultured in their cells, they're then removed from their cells and are cut into smaller fragments via restriction enzymes. Different restriction enzymes are used to produce different fragments. The BACs contain the same section of the genome
2. The fragments are then separated via electrophoresis
3. Each fragment is then sequenced using an automated process
4. Computer programmes then compare the overlapping fragments (due to the different restriction enzymes) in order to reassemble the whole BAC segment sequence.
102
What is a BAC?
It's a man made chromosome (so all the base pairs are known) that can be grown inside bacterial cells
103
Define comparative gene mapping
When you know the sequence of bases in a gene and you compare that to the same/similar gene in a different organism. They both code for the same/similar polypeptide
104
What are the 5 applications (the purposes) of competitive gene mapping? Aka comparing genomes.
1. It allows you to identify whether certain genes or proteins are present in all organisms which gives us clues to its relative importance to life. (if every organism has it, it must be essential)
2. It allows us to see evolutionary relationships, the more DNA sequences the organism shares, the more closely related they are
3. It allows us to see the effects of changes like mutations
4. It allows us to compare pathogens and similar non pathogenic organisms to identify what causes the disease allowing us to create drug treatments
5. It allows us to compare the DNA of individuals to find mutant alleles that increase the risk of diseases like cancer.
105
Once the sections of genomes have been placed into BACs, what are the four stages of sequencing?
1. A number of specific BACs are firstly cultured in their cells, they're then removed from their cells and are cut into smaller fragments via restriction enzymes. Different restriction enzymes are used to produce different fragments. The BACs contain the same section of the genome
2. The fragments are then separated via electrophoresis
3. Each fragment is then sequenced using an automated process
4. Computer programmes then compare the overlapping fragments (due to the different restriction enzymes) in order to reassemble the whole BAC segment sequence.
106
What is a BAC?
It's a man made chromosome (so all the base pairs are known) that can be grown inside bacterial cells
107
Define comparative gene mapping
When you know the sequence of bases in a gene and you compare that to the same/similar gene in a different organism. They both code for the same/similar polypeptide
108
What are the 5 applications (the purposes) of comparative gene mapping? Aka comparing genomes.
1. It allows you to identify whether certain genes or proteins are present in all organisms which gives us clues to its relative importance to life. (if every organism has it, it must be essential)
2. It allows us to see evolutionary relationships, the more DNA sequences the organism shares, the more closely related they are
3. It allows us to see the effects of changes like mutations
4. It allows us to compare pathogens and similar non pathogenic organisms to identify what causes the disease allowing us to create drug treatments
5. It allows us to compare the DNA of individuals to find mutant alleles that increase the risk of diseases like cancer.
109
What is a DNA probe?
A DNA probe is a short single stranded piece of DNA about 50-80 nucleotides long. It's complementary to the section of DNA that's being investigated.
110
A DNA probe is labelled so that it can be recognised. How is it labelled? 2 ways
1. A radioactive marker that can be revealed upon exposure to a photographic film
2. A fluorescent marker that emits a colour upon exposure to a UV light
111
Other than DNA probing, when are fluorescent markers used?
In automated DNA sequencing
112
Define electrophoresis
It's similar to chromatography and it involves the separation of different DNA fragments. All the fragments are negatively charged but the smaller fragments move through the gel towards the anode easier and therefore move more
113
Can copies of a DNA probe be added to any sample of DNA fragments? Explain
Yes it can because it's single stranded which means it can bind to any fragment where the complementary base sequence is present
114
When a DNA probe binds to its complementary strand, what is the process called?
Annealing
115
Define annealing
The binding via complementary base pairing
116
Why are DNA probes useful? 3 things
1. They can locate a specific gene that's wanted for genetic engineering
2. They can identify the same gene on a variety of different genomes from separate species
3. They candid entity the presence/absence of an allele for a particular genetic disease
117
Define primer
A short single stranded sequence of DNA around 10-20 base pairs in length.
118
When are primers used? Why are they used?
In sequencing reactions and polymerase chain reactions.
They are used because they can bind to a single stranded piece of DNA, unlike polymerase enzymes, which allows polymerase enzymes to bind to it and form a double strand.
119
Sum up a polymerase chain reaction (PCR) into 3 words
Artificial DNA replication
120
What is the purpose of a PCR?
| When is it used?
To generate multiple copies of a tiny sample of DNA.
It's used in forensic investigations where samples of DNA from a crime scene can be amplified to generate enough material for genetic profiling.
121
List 4 characteristics that DNA has which enables a PCR to occur.
1. It has antiparallel backbone strands
2. It's made of strands with a 5 prime end and a 3 prime end
3. It only grows from ab3 prime end
4. Base pairs pair up according to the complementary base pairing rules. AT CG
122
Describe 3 things that differentiates a PCR from natural DNA replication.
1. PCR can only replicate relatively short sections of DNA rather than entire chromosomes
2. Primers are required in order for the process to start
3. A cycle of heating and cooling is required in order to separate and bind strands whereas in natural replication, DNA helicase separates the strands
123
Describe the 4 steps of a PCR
1. The DNA sample is mixed with a supply of DNA nucleotides and DNA polymerase (which adds free nucleotides to the strand).
2. The mixture is heated to 95 degrees which breaks the hydrogen bonds that hold the 2 complementary DNA strands together. The samples are now single stranded
3. Primers are added and the temperature is reduced to 55 degrees. This allows the primers to bind via hydrogen bonds and form small sections of double stranded DNA at opposite ends of each sample.
4. DNA polymerase binds to these small sections of double stranded DNA and the temperature is raised to 72 degrees. This is the optimum temperature for DNA polymerase. The enzyme then adds free complementary nucleotides to the unwound DNA. DNA polymerase reaches the end of the strand and a new double stranded DNA is formed
124
Once a PCR has happened once, then what happens?
The process is repeated but this time, there are two double strands of DNA present, so it's like a snowball effect and each time it occurs more double strands are present and more are created. The amount of DNA increases exponentially
125
In a PCR what is the DNA polymerase enzyme described as?
It's described as thermophilic because it isn't denatured by extreme temperatures. The enzyme is derived from thermophilic bacterium called thermus aquaticus
126
I'm automated DNA sequencing, what does the reaction mixture contain?
DNA polymerase, many copies of a single stranded template DNA fragment, free DNA nucleotides and primers.
127
In automated DNA sequencing, what happens to some of the free DNA nucleotides?
Some of the free nucleotides are genetically modified to contain fluorescent markers so that when they are added to the growing strand, it throws DNA polymerase off the strand. This means no further nucleotides are added. Each nucleotide type is a different colour
128
Describe the 5 stages of automated DNA sequencing
1. The primer anneals to the 3 prime end of the template strand, this allows DNA polymerase to bind.
2. DNA polymerase adds free DNA nucleotides according to base pairing rules. This is why occurs in a PCR and in natural replication
3. If a modified nucleotide is added, DNA polymerase is thrown off the strand and the reaction stops.
4. As the reaction proceeds, many molecules/fragments of DNA are made at varying lengths. Sometimes the strand is completed, sometimes only one nucleotide is added
5. The strands then run through a machine which works like electrophoresis. A laser reads the colour sequence from the marked nucleotides, starting from the smallest strand to the largest. The sequence of couloirs and therefore bases can then be displayed.
129
What is the point of automated DNA sequencing?
It tells you the base pair sequence in a strand of DNA which helps us understand what sequences code for what proteins.
130
If people can't produce insulin, what type of diabetes do they have?
Type 1 diabetes mellitus
131
What are the disadvantages of using pig insulin for people with diabetes? 4 things.
How did they get the pig insulin?
1. It's not identical to human insulin and may get rejected
2. Less effective that bacterial insulin
3. It's very expensive
4. Only a small amount of insulin is present in the pancreatic tissue
It's extracted from pancreatic tissue
132
How many amino acids does insulin have?
How many base pairs are there in the DNA code? (Work it out)
Why is this a problem?
Only 51
About 153
This is a problem because it's very small making it hard to find in a genome which is massive.
133
How did they get the mRNA for insulin?
| From this, how did they synthesise a complementary DNA strand?
They use special centrifugation methods to separate mRNA of the right length from pancreatic tissue
They used an enzyme called reverse transcriptase in order to synthesis the DNA strand from mRNA, which gives us a copy of the template strand
134
Once they have synthesised a template strand for insulin from mRNA, what do they do next?
They mix DNA polymerase and a supply of DNA nucleotides with the single template strand. This means that the second strand in built using the template strand (the same as natural replication). This new double stranded DNA is called a cDNA gene.
135
After a cDNA gene has been synthesised from the mRNA for insulin, why happens next?
Unpaired nucleotides are added to each end of the cDNA, this creates sticky ends which are complementary to the sticky ends on the cut plasmid
136
After a cDNA gene has been given sticky ends, then what happens?
Plasmids are cut open using a restriction enzyme. The cut plasmids are then mixed with the cDNA and some of the plasmids take up the gene. DNA ligase then seals up the plasmids which forms recombinant plasmids
137
After recombinant plasmids containing cDNA are made, then what happens?
The plasmids are mixed with bacteria (E. coli) and some of the bacteria take up the recombinant plasmids to form transgenic transformed bacteria. All of the bacteria are then grown on an agar plate where each bacterial cell produces a colony of identical cloned cells.
138
When a colony is made from bacteria that has been mixed with the recombinant plasmids, why are the 3 types of colony?
Colonies from bacteria that didn't take up the plasmid
Colonies from bacteria that took up the plasmid, except the plasmid isn't recombinant as it didn't take up the cDNA
Colonies that took up the recombinant plasmid to produce transformed bacteria
139
Once the three types of colonies have been grown that could contain the recombinant plasmid, what are the genetic markers that help scientists identify the colonies?
The plasmids used originally are a vector and they contain resistance genes to the antibiotics ampicillin and tetracycline. The resistance genes are genetic markers and E. coli is susceptible to both antibiotic chemicals
140
How do scientists use genetic markers to identify which colony of bacteria is which?
Before anything happens, the plasmids are cut via restriction enzymes and the restriction site for tetracycline resistance is removed, this means that if a plasmid takes up the cDNA gene, it will break up tetracycline and the plasmid will no longer be resistant. However if it doesn't take up the gene, then it will reseal and the resistance will still work. All of the plasmids will still be resistant to ampicillin though.
141
Describe the procedure for replica plating. 4 steps
1. All of the bacteria are grown on a standard nutrient agar and they all form colonies
2. Some cells from each colony are transferred onto agar that's been made with ampicillin. This means that only bacteria that has taken up a plasmid will grow.
3. Some of the cells that have grown are then transferred onto an agar made with tetracycline. This means that only the bacteria that have taken up a plasmid without cDNA will grow.
4. From this we know that the bacteria that grew in the second agar but not the third is the one with the recombinant plasmid. This allows us to identify the colonies and grow then on a large scale which produces a lot of insulin.
142
Define golden rice
A biofortified rice that contains higher than normal concentrations of beta carotene
143
Many people die due to vitamin A deficiency, what continent is most at risk?
What people are most at risk?
The WHO believes that Africa is most at risk and some of south East Asia.
Children and pregnant women
144
Why is Africa most at risk against vitamin A deficiency?
Because it has many economically less developed countries where the populations can't obtain a balanced diet.
145
What food does poorer populations of Africa rely on?
Rice
146
What is another name for vitamin A?
How do you obtain vitamin A?
What properties does vitamin A have?
What does this mean?
Retinal or pro-vitamin A
From animal sources or from beta carotene which is a precursor substance that's converted to vitamin A in the gut.
It's fat soluble
It means that you also need lipids in your diet to absorb vitamin A
147
Name 4 functions of vitamin A
1. It forms part of the visual pigment rhodopsin for eyesight
2. It's involved in the synthesis of glycoproteins
3. It's needed for the maintenance an differentiation of epithelial cells, which reduces the risk of infection
4. It's essential for the growth of bones
148
Define sticky end
When DNA is cut using a restriction enzyme. It consists of a short run of unpaired, exposed bases at the end of a cut section and complementary sticky ends can anneal to recombine DNA fragments
149
What is another term for genetic engineering? Explain
Recombinant DNA technology. It's called this because the process involves combining DNA from different organisms in a single organism.
150
What are the 4 necessary steps for genetic engineering?
1. Obtaining the required gene
2. Placing a copy of the gene in a vector (a non-living thing)
3. Carrying the gene to the recipient cell via the vector
4. Getting the recipient to express the gene via protein synthesis
151
List 3 methods used in order to obtain the required gene for genetic engineering
1. The mRNA for that gene can be obtained from a cell that manufactures it. For example, the mRNA for insulin is obtained from beta cells. The mRNA can then be used as a template
2. The required gene can be synthesised via an automated polynucleotide sequencer
3. A DNA probe can locate the gene from DNA fragments and the gene can be cut from the DNA using restriction enzymes.
152
List 3 methods used in order to place a copy of a gene into a vector for genetic engineering
1. The gene can be sealed in a bacterial plasmid using DNA ligase (the most common method).
2. Genes can be sealed into virus genomes or yeast chromosomes
3. The inserted gene is transcribed in the host cell via the vector due to its regulatory sequences
153
List the 5 methods used in order to get the gene into the recipient cell for genetic engineering. The gene, once packaged in a vector, forms a large molecule that can't easily cross the membrane.
These methods aim to get the vector into the cell:
1. Electroporation is when a high voltage pulse is applied to disturb the membrane
2. Microinjection is when the DNA is injected into the host cell nucleus using a micropipette
3. Viral transfer is when the vector is a virus and it uses its mechanisms to infect cells by inserting DNA directly
4. Ti plasmids which are vectors that can be inserted into Agrobacterium tumefaciens (soil bacterium) and the plants can then be infected with bacteria, which inserts its DNA into the genome
5. Liposomes which are DNA molecules wrapped in lipid molecules, making them fat soluble which allows them to pass across the membrane.
154
What do recombinant DNA techniques usually involve?
| Give an example
Cutting and sticking DNA strands together.
| Example: Cutting out a useful chromosome and sealing it in a plasmid vector
155
What is the full name for restriction enzymes?
| Give an example of one
Restriction endonuclease enzymes
| EcoR1
156
What do restriction enzymes do?
How are they obtained?
How many different common restriction enzymes are there?
They cut through DNA at specific points where a specific sequence of bases occurs
They are extracted from bacterial cells that use them as a natural defence against viruses.
Over 50
157
Restriction enzymes cut through DNA at specific points, what are theses specific points called?
Restriction sites and they are less than 10 bases long
158
How does a restriction enzyme cut through a section of DNA?
It catalyses a hydrolysis reaction which breaks the phosphate-sugar backbone of DNA double helix in DIFFERENT places, resulting in a staggered cut/sticky end.
159
What enzyme joins complementary sticky ends together?
| How does it join them?
DNA ligase
| It catalyses a condensation reaction which joins the phosphate-sugar backbone of the double helix.
160
What other process is DNA ligase used in? Explain
Its used in natural DNA replication to join nucleotides together to form a DNA strand
161
In recombinant DNA technology, what is required in order for DNA ligase to join 2 sticky ends together?
It is required that the same restriction enzyme is used to cut the DNA fragments from different sources as it ensures the sticky ends are complementary to each other.
162
What is the DNA called once two complementary sticky ends have been joined together?
Recombinant DNA
163
How does genetic engineering use recombinant DNA?
It uses the DNA to create recombinant organisms described as transgenic. They synthesis useful products, e.g. insulin from E.coli
164
Define transgenic
An organism that contains an allele of a gene in its cells from an unrelated organism due to genetic engineering.
165
What are the 2 main reasons fro genetic engineering?
1. To improve a feature of the recipient organism
| 2. To engineer an organism that can synthesis useful products
166
Give 2 examples genetic engineering when a recipient organism is modified/improved.
1. Inserting a herbicide resistant gene into plants allowing the farmer to use herbicides on unwanted plants
2. Inserting a myostatin gene into livestock which promotes muscle growth
167
Give 3 examples of genetic engineering when an organism is modified to produce useful products
1. Inserting an insulin gene or growth hormone into bacteria and then growing them into large quantities for human use.
2. Inserting a gene for a pharmaceutical chemical into female sheep so that the chemical is produced in their milk.
3. Inserting genes for beta-carotene into the edible part of rice, this gene can then be turned into vitamin A
168
In genetic engineering, what is usually used as a vector?
A bacterial plasmid
169
What is a plasmid?
A small circular piece of DNA found in many bacteria, they are separate from the main bacterial chromosome. They often carry genes that code for the resistance to antibiotics chemicals
170
What happens if you mix quantities of cut plasmid and cut gene with ligase enzymes?
Some of the plasmids will combine with gene, sealing the gene in the plasmid. This produces recombinant plasmids.
Whereas some of the plasmids will simply reseal using ligase without any gene involved.
171
What happens after you have recombinant plasmids?
The plasmids are mixed with bacterial cells and some of the bacteria take up the recombinant plasmid
172
What can you do to increase the rate that bacteria takes up recombinant plasmids? 2 things
1. Adding calcium salts
| 2. Heat shock, this is when you lower the temperature of the culture to freezing then quickly raise it to 40 degrees.
173
Is the process of bacteria taking up recombinant plasmids efficient or not?
It is very inefficient
174
When bacteria take up recombinant plasmids, what is the bacteria then called?
Transformed bacteria which is transgenic as it contains new DNA.
175
Define conjugation
| What organisms can conjugate?
When genetic material is exchanged between organisms
| Bacteria
176
How does conjugation occur?
Copies of a plasmid are passed between bacteria of the same or different species via a tube.
177
Why is conjugation a concern?
Because plasmids are exchanged and this is where resistance to antibiotics are found. This means that the resistance can be passed between organisms; spreading the resistance quickly.
178
Give an example of a resistant strain of bacteria
Where is this bacteria found?
What problems can it cause?
MRSA
On the skin
If it gets into a wound it can seriously infect it
179
How is conjugation advantageous for bacteria?
Because it contributes to genetic variation and in terms of antibiotic resistance, it increases the chance of survival.
180
Define replica plating
The process of growing bacteria on an agar plate, then transferring a replica of that growth onto other plates which contain different growth promoters/inhibitors. The growth patterns on the different plates can then be analysed
181
What is the Latin name for rice plants?
Oryza sativa
182
Where is beta carotene found and why?
| Why is this a problem?
It's found in the green parts of the plant because it's a photosynthetic pigment
This is a problem because the part of plant that is eaten, the endosperm, has genes for beta carotene but they're switched off
183
Because the endosperm of rice plants doesn't contain beta carotene, what did scientists do?
They engineered rice plants so that beta carotene accumulated in the endosperm, this makes the rice a golden colour. Hence why the modified rice is called golden rice
184
To originally manufacture golden rice, normal rice needed to be genetically modified. How did they genetically modify rice to have beta carotene in the endosperm?
The enzymes needed for the production of beta carotene were already present in the endosperm. In order to activate the metabolic pathway that produces beta carotene, 2 genes needed to be inserted into the endosperm. The genes coded for phytoene synthetase which is found in daffodil plants and crt 1 enzyme found in soil bacterium. These were added near a specific promoter sequence that switches on genes for endosperm development. So the genes were expressed as the endosperm grew.
185
What is a disadvantage of golden rice?
It doesn't contain sufficient beta carotene so someone would need to eat large amounts to have sufficient amounts in their diet.
186
Once golden rice was manufactured, how did they try and improve the beta carotene yield?
They cross bred golden rice with natural rice varieties which produced 3-4 times more beta carotene
187
What happened after golden rice was made? (Talk about golden rice 2)
Golden rice 2 was made which had 20 times more beta carotene, this was a sufficient amount
188
Do farmers need a license to grow golden rice?
No because they offered humanitarian use licenses free of charge
189
Name 2 companies that oppose against golden rice
| Why are they against the idea?
Greenpeace and friends of the earth
Because it gains public acceptance for genetically modified crops
It reduces biodiversity
The safety of the rice is unknown
Genetically modified rice could breed with wild rice and contaminate their populations
190
Define genetically modified organism/GMO
An organism that has undergone genetic engineering
191
Define liposomes
Small spheres of lipid bilayer containing a functioning allele. They can pass through the lipid bilayer and thy can therefore act as vectors
192
Define gene therapy
Using molecular genetic technology to treat a disorder
193
How does gene therapy work?
It aims to get a working copy of a gene into cells that contain only dysfunctional copies. Transcription then occurs and the sufferers symptoms go away
194
Describe gene therapy in terms of RNA and give an example of a disorder it can help treat.
They could use interference RNA which can silence genes by binding to mRNA.
This treats cytomegalovirus infections in AIDS patients by blocking the replication of cytomegalovirus
195
Define somatic
A normal body cell
196
Finish the sentence
| Specialised cells all have a full genome but...
...certain genes are switched on/off so relatively few of them are active
197
What are the two types of somatic cell gene therapy?
Augmentation (adding genes)
| Gene therapy by killing specific cells
198
How does augmentation occur?
Some conditions are caused by the inheritance of a faulty allele leaving to the loss of a functional polypeptide. Augmentation engineers a functioning copy of the gene and inserts it into the specialised cell, this means a functioning polypeptide is synthesised
199
What is the process of gene therapy by killing specific cells?
This can treat cancers by eliminating populations of cells. The process involves using genetic techniques to make the cancerous cells express genes that produce surface antigens. This makes them vulnerable to attack by the immune system. This is a targeted cancer treatment
200
Why are the two types of gene therapy?
Somatic cell gene therapy
| Germline gene therapy
201
In a early embryo, what are all the cell types? 2 names for the same thing.
Totipotent stem cells that can become specialised.
| They're also called germline cells
202
True or false
| Each cell in a early embryo could become a new being
True
203
How does germline gene therapy work?
A gene is engineered into a sperm, egg, zygote or all cells in an early embryo which means the engineered gene will be present in every one of the organism's cells and it can function if the protein is required. The gene can also be passed onto offspring.
204
What are the ethical issues of germline gene therapy?
The new genetically modified gene could create a new disease or interfere with evolution
Permanent modifications could raise moral, ethical and social issues.
205
In gene therapy, are genes added or replaced?
| Why does gene therapy only woke on recessive conditions?
They are added
It only works on recessive conditions because genes are added and not replaced so if the disease is dominant then it will override the added gene.
206
Compare somatic cell gene therapy with germline gene therapy. 4 for each.
Somatic: delivery techniques are complicated because the location is needed, specific cells need to be removed and replaced etc, the treatment is short lived because specialised cells containing the allele won't divide, a vector is needed but hosts become immune to the genetically modified viruses and liposomes are inefficient, genetic manipulations are restricted to the patient.
Germline: delivery techniques are straightforward; the allele is introduced to germline cells, all cells and even offspring cells can contain a copy of the allele, tampering with human embryos is unethical and it could damage the embryo, genetic manipulations can be passed on to offspring
207
Define xenotransplantation
The transplantation of cell tissues or organs between animals of different species
208
Define allotransplantation
The transplantation of cell tissues or organs between animals of the same species
209
There is a worldwide organ shortage, how many patients on the waiting list die?
60%
210
What is one of the disadvantages of transplants?
| How can you resolve this disadvantage?
The transplanted organs are non self tissue which can trigger an immune response resulting in rejection
Make sure the organs are compatible and give the patient immuno suppressant drugs
211
Can xenotransplantation occur with humans
Yes
212
Why animal is usury used in xenotransplantation for humans?
Pigs
213
What can be done to reduce the likelihood of rejection before xenotransplantation with humans and pigs?
Engineer the pigs to lack alpha 1,3 transferase which is a key trigger in rejection in humans. You can also engineer human nucleotidase into pig cells which reduces the activities of immune cell activities involved in rejection
214
List 3 physiological problems in xenotransplantation in pigs
| List 3 ethical issues as well
1. Differences in organ size
2. Different lifespans so the pig organ may die prematurely
3. Body temperature in pigs is 39 degrees
1. Animal welfare groups strongly oppose
2. Against religious beliefs (pork)
3. Medical concerns/transmission of diseases from pigs
215
Name one benefit and 2 risks of using microorganisms for genetic engineering
Benefit: genetically engineered microorganisms produce useful products like human insulin
Risks: engineered microorganisms may escape from the contaminant and transfer their genes to other pathogenic organisms, genetic engineering uses antibiotic resistance as genetic markers this resistance could be passed on leading to a widespread resistance
216
Name 3 advantages and 5 risks of using plants for genetic engineering
Advantages: accumulation of beta carotene in rice endosperm could combat vitamin A deficiency, resistance to pesticides allows the application of weed killers which increases yield, pest resistance increases yield
Risks: the genetically engineered plants may pass on their genes to wild plants which reduces variation, the herbicide and pest resistance could be passed on to weeds resulting in super weeds, genes for pest resistance can be passed on to wild plants which disturbs the food chain, modified plants may be toxic, pathogen resistance could speed up evolution in pathogens
217
Name 3 advantages and 2 risks of using animals for genetic engineering
Advantages: pharmaceutical chemicals can be produced in milk, you can increase milk and meat production, you can improve the compatibility of xenotransplantation
Risks: animal welfare issues as there may be suffering, religious views (cows are sacred to Hindus)
218
Give 1 advantage and 3 disadvantages of using humans for genetic engineering (the problems are due to germline)
Advantage: gene therapy can treat some disorders
Risks: the effects of gene transfer can be unpredictable and could affect the embryo, no consent can be gained from the individual, germline gene therapy could be used to enhance favourable characteristics resulting in designer children
219
Is germline gene therapy used on humans?
No, it's illegal
