Chapter 21 Genetic Technologies Flashcards
1
Q
What does the genome refer to?
A
The genome is all of the genetic material of an organism or a cell. This includes coding and non-coding regions.
2
Q
Why are many individuals from a species used when carrying out genome sequencing?
A
More than one individual is used to create the reference genome as one organism may have anomalies/mutations in their DNA sequence that are atypical of the species.
3
Q
What is the proteome?
A
The proteome is all the proteins that an organism or a cell can produce.
4
Q
Why is the proteome larger than the genome?
A
Alternative splicing results in multiple proteins from one gene.
Post-translational modifications can result in multiple functional proteins from genes.
5
Q
Why is using the genome to determine the proteome difficult in complex organisms?
A
The presence of non-coding DNA and of regulatory genes means knowledge of the genome cannot be translated into the proteome,
6
Q
What is the epigenome?
A
The epigenome is the inherited changes in DNA that do not involve a change in DNA base sequence.
7
Q
Which organisms were prioritised to be sequenced? (give 5)
A
Humans
Model organisms such as drosophila and nematodes.
Organisms used in research such as mice.
Pathogens such as viruses and bacteria and parasites such as Plasmodium falciparum which caused malaria.
Pest species.
8
Q
How are genomes sequenced?
A
An automated process is used and sequencing methods are continuously being updated (you will be given detail in a question - you do not need to memorise methods).
9
Q
What can the data from genome sequencing be used for? (give 4)
A
To determine relatedness between species.
To identify mutations.
To identify potential antigens so vaccines can be produced.
To determine the code for spike proteins so that antibodies can be produced.
10
Q
What is meant by the term ‘recombinant DNA technology’?
A
Transferring DNA fragments from one organism or species to another.
11
Q
Why does recombinant DNA technology work so well?
A
The genetic code is universal as are the processes transcription and translation mechanisms.
12
Q
What does universal mean with respect to DNA?
A
The DNA triplet code for an amino acid is the same for all organisms.
13
Q
What other processes are described as ‘universal’?
A
Transcription and translation are also described as universal because all organisms use the same processes.
14
Q
What are the 3 main ways that DNA fragments are obtained?
A
Make a copy of the section you want (cDNA) from mRNA.
Use restriction endonucleases to cut the section you want from the DNA.
Build a fragment from nucleotides using a machine called a ‘gene machine’.
15
Q
What are restriction endonucleases?
A
Restriction endonucleases are enzymes capable of cutting DNA at a specific short sequence. For example Eco RI cuts the DNA at GAATTC.
16
Q
What is a ‘gene machine’?
A
A machine that uses a genetic code that has been inputted to build a gene fragment one nucleotide at a time.
17
Q
What are 2 uses for DNA fragments?
A
DNA fingerprinting
Genetic engineering.
18
Q
Via which two methods can DNA be amplified?
A
In vivo
In vitro
19
Q
What does in vivo mean?
A
Carried out inside a living body.
20
Q
What does in vitro mean?
A
Carried out outside of a living body e.g. in a test tube.
21
Q
What does DNA amplification mean?
A
Making copies of DNA.
22
Q
What does PCR stand for?
A
Polymerase chain reaction
23
Q
Is PCR in vivo or in vitro?
A
In vitro
24
Q
What are restriction endonucleases?
A
Enzymes capable of cutting DNA at specific sequences.
Examples:
25
When obtaining DNA fragments using restriction enzymes what must be added to the start and end of the desired gene?
Promoter and terminator regions.
26
When obtaining DNA fragments using restriction enzymes what is the name of the item that transports the desired gene into the host cell?
Vector (plasmids for bacterial host cells)
27
What does DNA ligase do?
When obtaining DNA fragments using restriction enzymes, DNA ligase must be used to bind the phosphate-sugar framework of the desired gene into the DNA with a phosphodiester bond (preferably using 'sticky ends').
28
What is the name given to DNA which consists of genetic material from two different organisms?
Recombinant DNA
29
What is the name given to an organism which contains genetic material from two different organisms?
GMO (genetically modified organism), transgenic or transformed. It has genetic information from more than one source.
30
What are the stages involved in making a GMO?
Identification
Isolation
Multiplication
Transfer
Identification and cloning
31
Describe what happens during the identification stage.
Bioinformatics is used to use the code for the gene to locate it in the genome.
32
When making a GMO explain what happens in the isolation stage.
The gene is isolated using 1 of 2 methods:
Reverse transcriptase is used to obtain DNA from mRNA.
Restriction endonuclease enzymes are used to cut the desired gene from the DNA
33
Where does the reverse transcriptase used in obtaining a DNA fragment come from?
Retroviruses are human viruses of which human immunodeficiency virus (HIV) is the best known.They have RNA as their genetic information but they can synthesise DNA from the RNA using reverse transcriptase (RT) (an enzyme).
34
When making a GMO explain what happens in the multiplication stage.
The desired gene is amplified (copied) using PCR (polymerase chain reaction).
35
When making a GMO explain how recombinant plasmids are made.
Restriction endonucleases cut at specific recognition sites to make either blunt or sticky ends. Sticky ends are better as there are exposed bases allowing hydrogen bonding to complementary bases to take place, as well as the phosphate-sugar phosphodiester bond made by the enzyme DNA ligase.
Promoter and terminator bases sequences are also added to initiate start and end of the gene being transcribed.
The desired gene is then inserted into a vector (most commonly a plasmid) which has had its DNA cut with the same restriction endonuclease so that there is a palindrome.
36
What are selectable markers?
Selectable markers are genes that are added to the plasmid vector so later it can be determined which plasmids have taken up the desired gene.
37
Give 2 selectable markers used in genetic engineering.
1 - UV fluorescence
2 - Antibiotic resistance
38
How can plasmids be inserted into host cells?
Calcium ions, temperature changes or electric shocks are used to make the membrane permeable to allow the plasmid vector to pass into the cell.
39
Why are all of the vectors not successfully taken up by the host cell?
Only some bacterial cells take up the plasmid vector.
Sometimes multiple fragments join together to form their own plasmids.
40
When making a GMO explain what happens in the last identification stage when the cells that have taken up the vector are identified.
Marker genes are used to identify which vector has successfully taken up the desired gene, and which host cell has successfully taken up the vector. Markers used are antibiotic resistance, enzymes and fluorescence.
41
Describe how antibiotics are used in the last identification stage in making a genetically modified organism.
The bacterial culture containing transformed and potentially not transformed cells is plated onto a medium containing an antibiotic. Only the transformed cells containing the gene for antibiotic resistance will grow. These cultures can be subcultured to make lots of copies.
42
Describe how fluorescence is used in the final identification stage of the production of genetically modified yeast.
A gene from a jellyfish plasmid which produces a green fluorescent protein (GFP) is incorporated into the vector.
The desired gene to be cloned is transferred to the centre of the GFP gene so any bacterial cells which have taken up the desired gene will not express the GFP so they will not fluoresce whilst the other cells without the desired gene will.
There is no need for replica plating as the desired cells have not been harmed.
All that is required is to retain the cells which do not fluoresce by viewing them under a microscope.
43
Why do bacterial cells have restriction endonuclease enzymes naturally?
Bacteria are often invaded by viruses (bacteriophages) which inject foreign DNA into their cells and so the bacteria have enzymes which cut up the viral DNA. These are RESTRICTION ENDONUCLEASES (RE). There are many different RE’s. Each cuts the DNA at a different sequence of bases called the recognition sequence.
44
What is another name for the recognition site that the restriction endonuclease cut at?
Cleavage site
45
Once all of the stages for producing a GMO with recombinant DNA have been completed, there is only one thing left to do. Grow or clone the new organisms. If your GMO is a bacteria what must you do to ensure maximum growth?
Ensure the conditions for bacterial growth are optimum (e.g. suitable temperature, water availability, Oxygen and a nutrient source).
46
Before we started to use gene technology to create new organisms, how did breeders and farmers alter their livestock?
Selective breeding over many generations.
47
Give 7 uses of DNA technology
Increased yield of farm animals and crops.
Improved nutrient content of food.
Introducing disease and pest resistance to animals and plants.
Making crop plants herbicide tolerant.
Developing tolerance to environmental conditions.
Making vaccines.
Producing medicines and treatment of disease.
48
What 5 items/apparatus are needed to perform PCR?
The DNA fragment to be copied.
DNA polymerase to join together the nucleotides in a hot environment.
Primers with short, set nucleotides complementary to those on the ends of the fragment to join to the ends of the DNA fragment. 15-20 bases long.
Nucleotides of all ATC and G to bind to complementary bases.
Thermocycler to control temperature changes during PCR.
49
What temperature is needed to separate the DNA strands in PCR?
Heat the contents of the thermocycler to 95°C to separate the DNA strands.
50
What temperature is needed for primers to anneal in PCR?
Cool the thermocycler contents to 55°C. This causes the annealing primers to attach (anneal) to the start and end complementary bases on the DNA strands. (this also keeps the strands apart).
51
What temperature is needed for DNA to bind new nucleotides in PCR?
Heat up to 72°C. This is the optimum temperature for DNA polymerase to cause new nucleotides to bind to their complementary base pairs.
52
Why do we use PCR?
PCR is the basis of Genetic Fingerprinting, where a sample from a known source can be compared against a sample.It allows even a tiny sample of skin, blood, a hair follicle or semen to contain enough DNA to be amplified in PCR.
53
Why is a thermocycler used in PCR?
PCR uses a computer controlled machine which allows temperatures to be controlled and timed accurately. The whole temperature cycle takes around 2 minutes so over 1 million copies of the DNA can be made in 25 cycles, and 100 billion can be made in a few hours.
54
Where do we get thermostable enzymes from to use in PCR?
Thermostable enzymes are from thermophilic bacteria (e.g. Thermus aquaticus) which survive in hot springs which join nucleotides together. We call it Taq polymerase.
55
What are the advantages of in vivo cloning?
In vivo cloning can add a gene to another organism via vectors and plasmids.
There is a low risk of contamination as the same RE is used to cut the same codes for all DNA involved so no stray DNA is present.
Only the desired genes are cut out.
This produces transformed bacteria to produce many products (e.g. insulin).
No prior knowledge of base sequences is needed.
56
What are the advantages of in vitro cloning?
- Quick. Can make billions of copies from a small sample in a few hours. - No living cells are needed.
57
What are primers and how are they used in PCR?
Short pieces of DNA with set bases complementary to those on the DNA sample. They attach to the start and end of the DNA to provide start and stop instructions for PCR. DNA polymerase can only attach nucleotides at the end of an existing chain. Primers also keep the 2 strands apart.
58
Why are 2 different primers needed in PCR?
The sequences at the 2 ends of the strand are different
59
What bond is broken when DNA strands are separated in PCR?
H Bonds
60
What is gene therapy?
The replacement of a faulty or defective gene with a functioning (or healthy) gene.
61
What are the two methods that can be used in gene therapy?
1 - Germ line therapy
2 - Somatic cell therapy
62
How does germ line gene therapy work?
Replacing or supplementing the defective gene in the fertilised egg. This means all of the developing cells will be normal, as will the cells of the next generation. The solution is permanently but currently prohibited for ethical reasons
63
How does somatic cell gene therapy work?
Targets the affected tissues (such as the lungs). This does not pass to the next generation as it is not in the gametes. The treatment is also short lived for the patient as the lung cells are continually dying and being replaced. The treatment therefore needs to be repeated every few days in some cases.
64
What is a DNA probe?
The DNA probe is a short, single stranded section of DNA that has an identifiable label attached to it.
65
What are the two types of DNA probes?
1 - Radioactively labelled probes
2 - Fluorescently labelled probes
66
How do radioactively labelled probes work?
Radioactively labelled probes are made of nucleotides with the isotope 32P. A photographic plate exposed to radioactivity to identify the probe.
67
How do fluorescently labelled probes work?
Fluorescently labelled probes emit light under certain conditions.
68
How do DNA probes result in a scientist being able to identify a mutant allele in a patient?
A probe with bases complementary to the bases on the portion of the gene we want to find is made.The DNA being tested is treated to separate the strands.The separated strands are mixed with the probe which binds the complementary bases on the strands. This is DNA hybridisation.The site at which the probe binds can be identified by radiation or fluorescence emitted.
69
What are labelled DNA probes used for?
Labelled DNA probes are used to screen patients for heritable conditions, drug responses or health risks.
70
What is the point of genetic screening?
Genetic screening is carried out on individuals who may carry the mutant gene, this is determined by looking at their family history. Screening can allow a couple to find out the chances of having a child with a genetic disorder. A couple at risk can then be referred to a genetic Counsellor for advice.
71
If a positive gene test result reveals that a seemingly healthy individual carries or has a genetic mutation associated with a specific disorder, what would a ‘negative gene test result’ be? How about a ‘false negative gene test result’?
A negative gene test result reveals that the faulty gene being looked for is not there. A false negative result means that further testing is probably needed as there was a problem with the procedure.
72
Why are introns (not exons) used in genetic fingerprinting?
Genetic fingerprinting is based on the fact that DNA the genome of any organism has many repetitive non-coding bases – INTRONS (e.g. 95% of human DNA does not code for any characteristic). Introns contain repetitive sequences of DNA called core sequences which differ in everyone (except identical twins).
73
What are the stages of DNA fingerprinting?
Extraction, Digestion, Separation, Hybridisation, Development
74
In DNA fingerprinting what happens in Extraction?
Extract DNA from the sample (however small) by separating it from the rest of the cell. PCR can be used to make more if necessary.
75
In DNA fingerprinting what happens in Digestion?
The DNA is cut into fragments using restriction endonucleases (chosen as they cut close to but not inside core sequences).
76
In DNA fingerprinting what happens in Separation?
Gel electrophoresis separates the DNA fragments according to size. The gel is then immersed in an alkali to separate the 2 DNA strands. The single strands are then transferred onto a nylon membrane by a technique called southern blotting
77
In DNA fingerprinting what happens in Hybridisation?
Labelled DNA probes with complementary sequences are used to bind to the core sequences. The temp and pH must be correct for binding to occur. Different probes are used to bind different core sequences.
78
In DNA fingerprinting what happens in Development?
An X-ray film is put over the nylon membrane and the film is exposed by the radiation in the probes (fluorescence probes are seen visually). A series of bars are revealed in a pattern of bars unique to the individual.
79
How does southern blotting work in the separation stage of DNA fingerprinting?
1 - A nylon membrane is laid over the gel and several sheets of absorbent paper are placed on top which draws up the liquid containing the DNA via capillary action.
2 - The DNA fragments are transferred to the nylon in the same position.
3 - The fragments are fixed in place on the membrane using UV light.
80
How are genetic fingerprint results interpreted?
A DNA fingerprint is compared for 2 or more samples of blood from the scene of a crime for example compared to the suspect(s). They are then visually checked. If there seems to be a match each fingerprint is passed through an automated scanning machine which calculates the length of the fragments from the bands (done via measuring the distance travelled by known lengths of DNA). Finally the odds are calculated of someone having an identical fingerprint . The closer the match between the two samples the more likely they came from the same person.
81
Give 8 uses of genetic fingerprints.
1 - Check immigration applications.
2 - Confirm animal pedigrees / ownership.
3 - Check evolutionary relationships.
4 - To determine genetic variability within a population.
5 - Paternity testing. Any bars which are not from mum will be from dad
6 - Animal and plant breeding
7 - Forensic science / solving crimes
8 - Medical diagnosis
82
What does VNTR stand for in genetic fingerprinting?
Variable Number Tandem Repeats
83
What is the probability of 2 individuals having the same VNTRs?
Very low.
