Molecular Genetics Flashcards
1
Q
What is DNA made from?
A
Deoxyribonucleotides.
2
Q
Which direction is DNA synthesised in?
A
5’ to 3’.
3
Q
How are DNA strands orientated?
A
Antiparallel.
4
Q
DNA melting curve - why does fluorescence decrease as temperature increases?
A
Because intercalating dyes (ethidium bromide, SybrGreen) only bind double stranded DNA.
5
Q
What is the melting temp. (Tm)?
A
The point at which there is 50% dsDNA and 50% ssDNA.
6
Q
How do GC rich sequences affect Tm and why?
A
They increase it because there are more triple H bonds.
7
Q
How do mismatches affect Tm and why?
A
They decrease it because mismatches are unstable and the DNA is therefore more easily denatured.
8
Q
What does Tm indicate?
A
The energy required to denature DNA.
9
Q
What does Tm depend on (3)?
A
- Proportion of A-T:G-C.
- Mismatching.
- Fragment length.
10
Q
How is Tm used?
A
To identify DNA variations: Alleles. Polymorphisms. Mutations. Contaminations/infections.
11
Q
How was cloning done before PCR?
A
Using cloning vectors with DNA fragments inserted and introducing them to host cells to grow into colonies.
12
Q
Why is PCR better than using cloning vectors?
A
It is quicker and easier, and cloning vectors often would not work.
13
Q
How does PCR work?
A
The DNA strands are denatured (95C), then primers bind to the fragments (60C) to initiate DNA synthesis and DNA polymerase builds the new strands by adding dNTPs to the 3’OH ends of the chains (72*C).
14
Q
What are the PCR reagents (5)?
A
- Template DNA.
- Primers.
- dNTPs.
- Buffer.
- DNA polymerase enzyme.
15
Q
What technology is used to carry out PCR?
A
A thermo-cycler.
16
Q
What do primers provide in PCR?
A
Specificity and directionality.
17
Q
What are the three stages of PCR?
A
- Denaturation.
- Annealing.
- Extension.
18
Q
What is electrophoresis used for?
A
PCR visualisation.
19
Q
What is used as a conductor in electrophoresis?
A
Ionic buffer.
20
Q
Which direction does DNA move in during electrophoresis?
A
Towards the positive electrode.
21
Q
How are PCR products visualised in electrophoresis?
A
Using intercalating fluorescent dyes.
22
Q
Why are reaction efficiency and accuracy lower when there are more cycles?
A
The reagents begin to run out as the amplification curve approaches the plateau. At this point, PCR is prone to errors.
23
Q
When is PCR amplification exponential?
A
When there is an abundance of reagents.
24
Q
What is an application of end point PCR?
A
Detection of size polymorphisms (fragments are separated by size in electrophoresis).
25
What are the benefits of end point PCR?
1. Sensitive (only 1 DNA molecule needed).
2. Specific (primers).
3. Fast.
4. Flexible (adapted for different investigative purposes).
26
What is the downside of end point PCR?
It is not quantitative.
27
How is qPCR/rtPCR different from end point PCR?
It visualises the amplification curve in real time and measures the relative and absolute levels of DNA.
28
How is qPCR used?
1. Gene expression.
2. Detecting bacterial contamination eg. of water.
3. Detecting food contamination (e.g. horse meat)/GMO.
4. Detecting contamination of crime scene DNA.
5. Human diagnostics/genetic screening.
6. Detecting infection/cancer progression.
29
How is rtPCR DNA visualised?
1. Intercalating dyes.
| 2. Fluorophore-linked oligonucleotide TaqMAN probe.
30
What is the limitation of using intercalating dyes to visualise rtPCR products?
Visualises all PCR products - specific and not. If primers are not specific, incorrect DNA will be shown.
31
How do TaqMAN probes work?
They contain a fluorescent dye and a quencher. When the 2 are separated by hydrolysis, light is visible.
32
How does the TaqMAN probe increase PCR specificity and how is it used?
Only specific amplicons will fluoresce. It can detect specific mutations and foreign DNA, and can be used in genotyping.
33
What are the advantages of using intercalating dyes over TaqMAN probes?
They are easier and cheaper.
34
Where are TaqMAN probes most used?
In clinical and environmental diagnostics.
35
How is fluorescence detected in rtPCR?
Using a photodetector.
36
What is fluorescence proportional to in rtPCR?
Amplified DNA.
37
What are the 2 main parameters given by qPCR?
1. Visualisation of amplification curves for quantification (accumulation of fluorescent signals gives amount).
2. DNA melting curve for product identification.
38
What is the cycle threshold (Ct)?
The cycle number at which you can detect what you're looking for. More target DNA at the start = a lower Ct value.
39
When there is more infection/contamination, what happens to the Ct value?
It shifts to the left.
40
What is analytical sensitivity?
How often a positive result is returned when the target is present.
41
What is analytical specificity?
How often a negative result is returned in the absence of the target.
42
In what areas does PCR have important applications?
1. Medicine.
2. Environment.
3. Forensics.
4. Evolution.
5. Research.
43
What is COLD PCR used for?
Preferentially amplifying rare targets/mutant sequences. The mutations do not need to be known. Good for early detection of cancer (low % mutant/malignant cells) and detecting resistance mutations (cancer/virology).
44
What is COLD PCR?
Co-amplification at lower denaturation temperature PCR.
45
How does COLD PCR work?
A few cycles are carried out then slow annealing leads to the formation of heteroduplexes (where mutant DNA strands hybridise with normal strands). These are mismatches so have a lower denaturation temperature. Continuing PCR using a the lower denaturation temperature results in the denaturation and further amplification of the heteroduplexes only, meaning mutant strands are selectively amplified (from initially very low % mutant DNA to 50%).
46
Why is the efficiency (speed) of rtPCR important?
It is used in diagnosis for early detection, where treatment must be decided quickly (infection) and spread must be prevented.
47
When is cDNA used?
For amplification when from starting from mRNA or virus RNA.
48
How is cDNA made?
Retrotranscriptase. A linear reaction.
49
Why is specificity of rtPCR important?
Better diagnosis and treatment - different drugs for different bacterial/viral strains.
50
What is serology?
Blood test to detect antibodies in the patient (measure immune response). Can be compared to pathogen (genome detected by rtPCR).
51
For pathogen detection, where should a specimen be taken from?
Wherever the pathogen is found for the particular disease e.g. lungs or lymph nodes.
52
What are rtPCR applications and case studies?
```
Infections:
1. Dengue fever (Aedes aegypti).
2. HIV.
3. Ebola.
4. Marburg haemorrhagic fever.
(All RNA viruses).
Environment:
1. Soil nitrification.
```
53
What are 4 mosquito borne viruses?
1. Dengue (4 serotypes).
2. West Nile.
3. Chikungunya.
4. Zika.
54
Where is Dengue found?
Countries in C/S America, Africa, Asia. Subtropical and highly populated but spreading further North into Europe (warming).
55
What are symptoms of dengue fever (febrile phase)?
Sudden onset fever, headache, nose/mouth bleeding, muscle/joint pain, vomiting and diarrhoea, rash.
56
What are symptoms of dengue fever critical phase (haemorrhagic fever)?
Hypotension, pleural effusion, ascites, GI bleeding.
57
What are symptoms of dengue fever recovery phase?
Altered consciousness, seizures, itching, slow heart rate.
58
How is rtPCR applied to dengue?
Early detection and intervention. Preventing epidemics. Identifying the strains to decide the best treatment and identify new mutations.
59
Serology and dengue:
Viraemia peaks in acute phase, antibodies vs virus rises gradually and peaks in convalescence. 2 samples are collected. Can't detect the different serotypes.
60
rtPCR and dengue:
DENV1-4 primers and TaqMAN probes amplify and identify viral DNA sequences. Uses cDNA. Inactivated virus as dengue control and RNase P as human control (human gene).
61
What are the stages of HIV infection?
1. Acute - high viraemia, flu like symptoms.
2. Asymptomatic but progressive.
3. AIDS - increasing viraemia, infections and proliferative disorders.
62
When is qPCR used for HIV patients?
1. At entry into care.
2. Every 3-6 months in untreated patients to monitor progression.
3. 2-8 weeks after starting HAART drug therapy.
4. Every 4-8 weeks until the viral load is below detectable limits.
5. 2-8 weeks after changing therapy.
6. Every 6-12 months to monitor effectiveness of therapy.
63
What are parameters of HIV infection?
1. CD4+ levels.
| 2. Viral load (viraemia).
64
What CD4+ levels show in HIV?
Decreasing CD4+ = worsening infection.
| Increasing CD4+ = improving infection.
65
What are the symptoms of Ebola?
Days 7-9 = headache, fatigue, fever, muscle soreness.
| Day 10 = sudden high fever, vomiting blood, passive behaviour.
66
How are IgM and IgG antibodies measured?
ELISA.
67
When does viraemia peak for Ebola?
With the onset of symptoms (day 7). Viraemia is a strong predictor of outcome.
68
How long after infection are IgM and IgG detected?
```
IgM = 3-6 months.
IgM = 3-5+ years (possibly lifelong).
```
69
When in the course of the disease is qPCR used?
Within days of onset of symptoms.
70
Why is analytical sensitivity critical for Ebola?
Early detection is important in preventing spread.
| Patient discharge is based on a -ve qPCR result.
71
What is nitrification?
Ammonium -> nitrite (nitrosomonas) -> nitrate (nitrobacter).
72
How is qPCR used for soil nitrification?
Analysis of amoA gene (key enzyme for oxidation of ammonia - present in bacteria and archaea) abundance and mRNA expression.
Comparing abundance and activity of bacterial and archaeal amoA genes (archaeal dominant over bacterial).
73
Issues in food security:
Need enough food to meet human demand, produced sustainably.
74
How is population growth disproportionate?
Africa -> 16-25% of global population by 2050. Major growth in Africa/Asia while in developed countries, population is stabilised.
75
Issues with too much food consumption:
Majority of population.
Half of all chronic diseases linked to poor diet.
Burden on national welfare services.
1% reduction in CVD -> £30m/yr saving for NHS.
3% of global GDP lost due to diet related disease.
High calorie food consumed but low in nutrients.
76
What is produced from oil refining?
Household goods - lubricants, waxes, polishes.
Petrochemical products - pharmaceuticals, industrial/specialty chemicals.
Asphalt.
Fuel.
77
Based on existing reserves, when will fossil fuels run out?
Oil - 50yrs.
Gas - 60yrs.
Coal - 120yrs.
78
How much of oil consumed globally is from fossil reserves as opposed to renewable resources?
97%.
79
How are biofuels and chemicals produced?
Fermentation of plant sugars.
80
What is the food vs fuel for land problem?
Land to grow food crops vs fuel crops.
Renewable oil production needs to triple in the next 20 years, meaning 3x more land used for fuel rather than food, or crop yield must be enhanced and oil content increased.
81
What are green revolution initiatives?
```
Increased agricultural production:
High yielding crop varieties.
Fertilisers.
Irrigation.
New (mechanised) cultivation methods.
=Tripled production and yield.
```
82
Issues for a second green revolution:
1. Restricted land use.
2. Competition with non-food agriculture.
3. Quality must be improved.
4. Chemical use and input resources (e.g. water) must be reduced.
5. Must be tolerant to erratic environmental conditions (climate change).
83
What are sources of genetic variation for crop improvement?
1. Genetic collections.
2. Wild populations.
3. Induced mutations.
4. Breeding.
5. Genetic manipulation.
May be closely related (primary), more distantly related (secondary), marginally related (tertiary), not at all related (quaternary - all organisms, transgenic by GM).
84
What is conventional breeding?
Selectively breeding for better traits. Uses natural variation. Has resulted in crops very different from their ancestors.
85
What is marker assisted selection?
Using molecular markers to assist selective breeding.
86
What are genebanks?
Maintain diversity from natural variation in tissue culture.
87
What is mutation breeding?
Creating random mutations using radiation or chemical mutagens. First released variety improved tobacco quality (Indonesia, 1934).
88
What are commonly used chemical mutagens?
1. Alkylating agents - EMS, MNU...
2. Sodium azide.
Also other chemicals.
Plant material is soaked in specific conc. for specific length of time.
89
What are commonly used physical mutagens?
Ionising radiation - gamma rays, X rays, fast neutron, ion beam.
Ultraviolet radiation.
Plant material is exposed for calculated amount of time to reach specific dose.
90
What types of mutations can chemical and physical mutagens cause?
```
Chemical:
Point mutations.
Insertions and deletions.
Physical:
Above.
Duplications.
Translocations.
Inversions.
```
91
Are more mutant crops produced by physical or chemical mutagens?
Physical.
92
What is an example of a mutant crop?
Calrose 76 semi-dwarf rice produced by gamma irradiation resulted in a 15% yield increase.
93
What are genetically modified organisms?
Organisms in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination.
94
What does GM do?
Produces transgenic plants. Foreign genes are introduced and expressed in plant cells to give the plant a new, useful characteristic.
95
What are differences between conventional breeding and GM?
```
Conventional:
A large amount of DNA is exchanged/introduced.
The DNA is not characterised.
DNA comes from closely related species only.
GM:
A small amount of DNA is introduced.
The DNA is well characterised.
DNA could come from any source.
Transformation/tissue culture process.
```
96
When we're GM plants first created?
Early 1980s.
97
What were the first plants genetically modified?
Tobacco, petunia and sunflower.
98
What are the steps of genetic modification?
1. Insertion of gene of interest into vector (T1 plasmid - A. tumefaciens, restriction and ligation).
2. Delivery of the vector into plant cells.
3. Moment of the DNA to the nucleus and integration into the genome.
4. Selection of transgenic cells.
5. Regeneration of those cells into transgenic plants.
6. Passed to future generations by Mendelian inheritance.
99
What is agrobacterium mediated transfer?
Infectious agrobacterium genes (rhizogenes?) produce virulence proteins - transport channel and packaging proteins.
T-DNA forms a complex with the packaging proteins and enters the host cell through the transport channel.
T-DNA enters the host cell nucleus where it is integrated into host DNA.
100
What does T-DNA of the Ti plasmid contain?
Oncogenic genes - auxin and cytokinin biosynthesis. Overproduction produces callus - crown gall disease
101
How is T-DNA modified to produce transformed plant cells?
Genes between T-DNA borders are replaced with genes of interest. Instead of tumours, transgenic cells are produced. Antibiotic resistance genes are included for selection.
102
How are whole transgenic plants grown from the transformed cells?
They are grown on media with auxin and cytokinin.
103
How are genes of interest/selectable markers expressed?
A promoter is included before the gene of interest. After there is a terminator. There may also be a transit peptide.
104
What types of promoters are there for modulation gene expression?
1. Constitutive promoters eg cauliflower mosaic virus promoter. Expressed in every tissue.
2. Organ and tissue specific promoters.
3. Inducible promoters.
4. Promoterless reporter gene constructs (to find new organ/tissue specific promoters).
105
What are the biological methods of delivering DNA into plant cells?
1. Agrobacterium.
2. Other bacteria.
3. Viruses.
106
What are the physical methods of delivering DNA into plant cells?
1. Particle bombardment.
2. Electroporation.
3. Silicon carbide whiskers.
4. Carbon nanofibres.
107
What particles are used in particle bombardment?
Gold or tungsten.
108
How do silicon carbide whiskers work?
Cells or tissues are vortexed with coated whiskers, which pierce the cells to introduce DNA. Commonly used with cereal crops.
109
What are the steps of plant tissue preparation and regeneration?
1. Plantlets are produced under sterile conditions.
2. Hypocotyls/cotyledons are prepared and co-incubated with Agrobacterium containing the transformation vector.
3. Cultivated on media containing kanamycin for selection.
4. Cultivation on cytokinin media for shoot development.
5. Cultivation on auxin media for root development.
6. Acclimation from tissue culture to soil.
7. Primary transformants produced.
110
How do you test if the DNA has been incorporated into the host plant genome?
Southern blot (DNA).
111
How do you test whether the GOI is expressed?
Northern blot (RNA) or qPCR.
112
How do you test whether the transcript of the GOI is translated?
Western blot (protein).
113
How do you test whether the GOI is inherited in a Mendelian fashion?
Segregation.
114
How can the trait produced by the GOI be observed?
May be seen in phenotype. Can test enzyme activity.
115
How would you test if the GOI trait is stable under different environmental conditions?
Field trials.
116
What is agroinfiltration?
Forcing transgenic agrobacterium into Nicotiana benthamiana leaves. Results in overexpression. Useful in producing therapeutic proteins eg. vaccines.
117
What is marker assisted selection (MAS)?
Using DNA markers closely linked to the target loci or alleles instead of/to assist phenotypic selection.
Assumes that DNA markers can reliably predict phenotype.
118
What molecular markers are used in MAS?
Can be an allele with a SNP or a gene close to the trait locus.
119
Is MAS transgenic?
No. Uses natural variation.
120
What are the advantages of MAS?
1. Simpler than phenotypic screening, may save time and resources.
2. Can select plants as seedlings, before transplanting/propagation, only plants with marker kept and grown. Important for grain/fruit quality traits.
3. More reliable, no environmental effects and can discriminate between heterozygotes and homozygotes.
121
How is MAS used?
Industrially, most crops for food, bioenergy and pharmaceuticals developed using MAS.
122
How is MAS carried out?
1. Plant tissues are sampled and DNA is extracted.
2. PCR and electrophoresis.
3. Marker analysis from electrophoresis.
123
What is the change in global distribution of biotech crops from 1996 to 2014?
There is a larger area in developing countries compared to industrial countries (a recent change).
124
Which country grows the highest % of GM crops?
The USA.
125
What is the difference in GM laws between Europe and USA?
GM testing is required in Europe, but in America GM foods are regarded as equivalent to natural food and not subject to FDA regulations (generally recognised as safe).
126
What are the main GM crops?
Soybeans - 82% of GM crops.
| 74% of soybeans modified for herbicide tolerance.
127
What are the 3 main companies selling GM crops?
1. Monsanto (US).
2. DuPont (US).
3. Syngenta (Switz.).
128
What are the benefits of GM?
1. Higher yields/productivity - food security.
2. Reduced pressure on renewable resources - biofuel crops.
3. Crops tolerant to climate change/fluctuations.
4. Crops grown on marginal soil/detoxify contaminated land.
5. Reduce the use of pesticides/herbicides.
6. Improve post-harvest properties - food security.
7. Produce pharmaceuticals more quickly and cheaply.
8. Improve nutritional quality.
9. Less soil erosion (no tilling).
10. Increased profits.
129
What are the risks of GM crops?
Environmental:
1. Unintended harm to other organisms (difficult to design pest specific toxins).
2. Reduced effectiveness of pesticides (resistance).
3. Gene transfer to non-target species (herbicide resistant superweeds).
Health:
Allergenicity?
Unknown effects?
(No deaths from GM foods, scientists do not believe they are a risk to human health).
130
What solution is there to the risks of GM crops?
Creating a buffer zone.
131
What are the economic concerns of GM crops?
1. Lengthy and costly process.
2. Industry monopolies/patented technologies for GM crops.
3. Unaffordable to farmers in developing countries.
4. Produce sterile seeds that do not germinate - only one growing season, must buy new seeds each year.
132
What are the laws on GM food labelling in Europe/Japan and US/Canada?
Mandatory in Europe and Japan, voluntary in US and Canada.
133
What is the Seralini rat tumour study?
Study that showed rats fed roundup ready corn (GM) over their lifetimes showed higher incidence of cancer and death than rats fed conventional corn. Is GM or roundup toxic (or both)?
134
What were the responses to the Seralini rat tumour study?
1. Study was criticised as it had a low sample size and used Sprague-Dawley rats which are susceptible to cancer - poor experimental design.
2. European food safety authority - study of insufficient scientific quality for safety assessments.
3. Researchers wouldn't release data - questions of scientific misconduct.
4. Paper retracted by the journal and republished in one without peer review.
135
Who is responsible for ensuring safety of GM foods?
1. European food safety authority (efsa).
2. US food and drug administration (FDA).
3. Food standard agency (UK).
4. US department of agriculture (usda).
5. Department for environmental, food and rural affairs (defra).
136
What do safety evaluations for GM foods require?
1. Environmental evaluation from repeated field trials in different locations.
2. Toxicology studies.
3. Substantial equivalence to the existing product or similar ones in the market based on comparison of molecular and biochemical characteristics. Toxicology studies carried out where components are identified as significantly different.
137
GM conclusions:
No technology is risk free, but GM is not especially risky.
Competing practices/technologies are also not risk free.
Benefits balanced against not using the technology.
GMOs can be used to improve agricultural and environmental security and sustainability.
138
What is CRISPR/Cas9?
Genome editing technology.
139
What are some examples of GM traits?
1. Herbicide tolerance.
2. Insect resistance.
3. Anti-allergy.
4. Delayed fruit ripening.
5. Stress tolerance.
6. Enhanced photosynthesis/yield.
7. Fertility/sterility.
140
What are input traits?
Traits that potentially alter inputs required in production.
| Most 1st gen GM crops have input traits.
141
What are output traits?
Traits that alter the harvested product, improving the quality.
142
What is an example of an input trait?
Glyphosate resistance.
The dominant herbicide worldwide, broad spectrum, highly effective.
Toxicologically and environmentally safe.
143
What does glyphosate do?
Inhibits EPSP synthase, preventing amino acid synthesis and resulting in death.
144
What are strategies to generate glyphosate resistance in crops?
1. Increase EPSP synth conc. to saturate the inhibitor.
2. Make EPSP synth. resistant to glyphosate.
3. Use Agrobacterium CP4 EPSP synth. resistant to glyphosate - most successful.
145
How do herbicide resistance GM crops increase yield?
The crop is unaffected by the herbicide while weeds are killed. No competition with weeds for nutrients.
146
What is an example of a herbicide resistant crop?
Roundup ready soybeans are resistant to roundup herbicide.
147
How are most insect resistant crops produced?
Using Cry gene from Bacillus thuringiensis which leads to production of a protein which causes paralysis and death to many insects.
148
How were GM papayas resistant to papaya ringspot virus developed?
University of Hawaii, 1980s. Virus resistance gene encoding viral capsid proteins that trigger an immune response introduced.
149
What is an example of a GM plant with an output trait?
Delayed ripening tomatoes - longer shelf life than conventional tomatoes.
150
How are delayed ripening tomatoes created?
An antisense mRNA (from synthetic antisense PG gene) silences the polygalactouronase gene transcript. PG is a cell wall degrading enzyme that leads to natural ripening/rotting. Stopping PG delays ripening by approx. 3 weeks.
151
What is an example of a GM crop with improved nutritional quality?
Golden rice - beta carotene (provitamin A).
152
Why is vitamin A deficiency a problem?
Mainly in Sub-Saharan Africa and SE Asia.
1/3 of children under 5 have VAD.
0.5 million children under 5 are blind due to VAD.
1 million children under 5 due from VAD.
153
What are the functions of vitamin A?
1. Vision.
2. Gene expression.
3. Reproduction.
4. Embryonic development.
5. Epithelial cell maintenance.
6. Growth and immune function.
154
What is beta carotene?
A carotenoid with beta rings which is a precursor of retinal (vitamin A).
155
How was golden rice produced?
The beta carotene biosynthetic pathway was engineered into rice endosperm. Rice naturally lacks 4 enzymes required to convert geranylgeranyl diphosphate into beta carotene.
156
What genes were added to the rice to produce golden rice?
1. A daffodil gene producing phytoene synthase function (geranyl... -> phytoene).
2. A bacterial gene providing carotene desaturation enzyme crtI
(phytoene -> lycopene).
3. A daffodil gene providing lycopene-beta-cyclase function
(Lycopene -> beta carotene).
157
Why is CrtI useful in the beta carotene biosynthetic pathway?
It is a single bacterial gene which acts as a substitute for 4 different plant genes in converting phytoene to lycopene. A useful shortcut.
158
How much carotenoid is produced per gram of dry golden rice?
35 micrograms (according to HPLC analysis).
159
How much vitamin A does 50g (uncooked) golden rice provide?
>90% of vit A requirement.
| >60% of RDA (which is higher).
160
What is molecular phylogenetics?
Studying DNA sequences (rather than taxonomic traits) to work out the evolutionary relationships within/between species.
161
Why is it important to understand evolutionary relationships?
1. Documenting evolution.
2. Forensics (relationships).
3. Health (understanding where a pathogen came from by what it is related to).
4. Conservation (how distinct are different populations?).
5. Wildlife forensics (which population a sample came from).
162
Which mutations are inherited?
Germline mutations.
163
What does the number of mutations separating two species indicate?
How recently they diverged.
164
What is used to construct evolutionary trees?
Molecular markers - small, homologous sections of the genome selected for comparison.
165
What type of marker is appropriate for fine-scale resolution (e.g. between bumble bee and honey bee)?
A fast evolving marker, because a slow evolving one won't have changed enough to provide any information.
166
What type of marker is appropriate for resolving more distant relationships (e.g. between bees and goats)?
A slow evolving marker, because a fast evolving one will have changed too much so that the 2 sequences may not be recognisable as homologues.
167
What is the rate of evolution?
The rate at which mutations accumulate.
168
What determines how fast a marker evolves?
1. The mutation rate (which differs between genome regions and species).
2. The effects of the mutations on the phenotype.
169
What is the main source of mutation in germline cells?
Replication errors.
170
How do replication errors occur?
An incorrect nucleotide may be inserted by DNA polymerase opposite a damaged base (10^-6 to 10^-9 errors per base pair per cycle). DNA repair enzymes miss approximately 1 in 10 errors, so each mammalian offspring differs by at least a few mutations from their parents.
171
What is the most common type of mutation (polymerase or environmentally induced)?
Point mutations - insertion, deletion or substitution of a base.
172
What are base substitutions?
Can be transversions - purine pyrimidine.
| Or transitions - A G or C T.
173
What are inversions?
The reversal of an entire sequence.
174
How large can insertions and deletions be?
They can involve several or even 100s of bps.
175
How does the impact of mutations on phenotype affect marker evolution?
In non-coding DNA, mutations don't affect phenotype, so the mutation rate is not affected by selection - fast marker evolution (rate = mutation rate).
In coding DNA, mutations can be synonymous (don't change the AA sequence), or non-synonymous. Synonymous mutations also don't affect phenotype so are not affected by selection, but non-synonymous mutations are usually deleterious and are therefore removed by selection (very few are beneficial) - slow marker evolution in coding regions.
176
What is organelle DNA?
rRNA or mtRNA.
177
What is nuclear DNA?
Microsatellites.
178
Why is rRNA useful in creating ancient phylogenies?
It is present in all cells and has the same function. Easy to align and compare.
179
Which gene codes for the part of a ribosome which is made of RNA?
The 16s RNA gene (16s DNA - coding). Ribosomal RNA is therefore also a phenotype.
180
Which parts of the 16s RNA gene evolve very slowly?
Parts that code for regions determining the shape of the molecule (as it must fit perfectly into the ribosome).
181
Why do parts of the 16s RNA gene which do not affect tertiary structure evolve more quickly?
Mutations in these regions are less likely to be deleterious.
182
How were archaea replaced based on 16S DNA?
Were previously placed within the bacteria, but 16s DNA showed they were actually as distinct from them as eukaryotes are.
183
Why is the rate of synonymous mutations fast in mtDNA?
Mitochondria have their own DNA and proofreading is poorer. May also have a higher mutation rate due to byproducts of respiration.
184
Why are species specific primers not required for amplification of mtDNA?
Arrangement of genes is conserved.
185
What is mtDNA used for?
Resolving more recent divergences, as it is relatively fast evolving. Some parts can be used to identify relatives within a species (maternal lines).
186
What are the key mtDNA markers?
1. Cytochrome b - coding, quite variable.
2. Control region - non coding, most variable.
3. Cytochrome oxidase 1 (cox1) - coding, quite variable.
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What is the animal DNA barcode region in mtDNA?
Cox1 - varies between species, but rarely within them.
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How are maternal lines traced using mtDNA?
Using a mtDNA marker that varies within species (not the barcode region).
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How has mtDNA been used in forensic identification?
Richard III was identified by mtDNA from a direct descendant of his mothers maternal line.
The last Russian imperial family (Romanovs) were identified by mtDNA which matched Prince Philip (a direct descendent from the maternal line).
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In phylogenetic trees, what do branch lengths and numbers indicate?
Branch lengths - amount of evolutionary change.
| Numbers - estimated time since divergence.
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How are dates assigned to divergences?
Using a molecular clock.
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What is the molecular clock hypothesis?
Nucleotide substitutions occur at a constant rate, so knowing that rate means we can tell how long ago 2 species diverged.
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How is the molecular clock calibrated using the fossil record?
(No. of substitutions/millions of years since divergence)/2 = substitutions per million years.
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Why can a universal molecular clock not be applied to all sequence data?
1. Synonymous mutations occur more quickly.
2. mtDNA evolves more quickly.
3. Generation times affect molecular clocks.
4. Molecular clocks appear to have increased in speed over the last 1-2ma as some deleterious mutations not yet removed.
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Why does retrovirus DNA evolve quickly?
Reverse transcriptase lacks proofreading ability.
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What is the transcriptome?
A collection of mRNA that indicates gene activity.
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Why is there differential gene expression in cells with the same genome?
Different genes are turned on or off. Expression reflects the functions of the cell, e.g. a gene expressed in fat tissue but not bone might be involved in lipid metabolism.
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How much of the genome is transcribed in to mRNA?
Less than 5% in humans.
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Transcriptomics steps:
1. Collecting samples and mRNA extraction.
2. Microarrays or RNAseq used to work out which genes the mRNA came from.
3. Quantify gene expression or compare transcriptomes for different phenotypes.
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Why are samples flash-frozen immediately for transcriptomics?
RNA is less stable than DNA and starts to degrade quickly after death. After this it cannot be sequenced.
For behavioural studies, samples flash frozen alive to show gene expression at the specific time the behaviour is displayed, as stress/death could change gene expression.
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What are microarrays?
Grids containing hundreds of spots of ssDNA (probes) each with a different sequence corresponding to one gene of interest.
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How do microarrays work?
Once mRNA has been extracted and converted to sscDNA (by reverse transcriptase, as cDNA is more stable), the cDNAs are labelled with fluorescent markers and the microarray is flooded with them. The sscDNAs hybridise with complementary ssDNA probes, and will be visible under UV light.
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How is gene expression quantified in a microarray?
Each spot contains up to 10^9 copies of the same probe (higher than the number of copies of any mRNA to prevent saturation). The amount of fluorescence shows how much mRNA there was.
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How are transcriptomes from different tissue samples compared (eg. from healthy and diseased plants)?
The different cDNA mixtures have different fluorescent markers (e.g. green or red). The samples are combined and washed over the microarray. The colour and amount of fluorescence at each spot shows whether mRNA is present in one or both transcriptomes, and how much.
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How are microarrays used in cancer (diffuse large B lymphomas - wbc cancer)?
Analysis shows that in healthy patients, germinal centre B cell genes are expressed when B cells are dividing, but not when they are resting. Suggests searching for these genes in tumours. Gene expression subtype in patients indicates survival prospects and what is going wrong (personalised medicine). Germinal centre B cell gene expression subtype shows higher survival than activated B cell gene expression subtype.
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How can microarrays be in animal behaviour studies?
Bee waggle dance - study showed slightly different gene expression profiles between 2 groups of bees which were communicating different foraging distances. The changes were in the region of the brain associated with learning and memory. 52 distance related genes were found using the microarray.
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What is the problem with hybridisation in microarrays?
Similar mRNAs might activate the same probe, as probes and cDNA do not have to be perfectly matched to hybridise. This means similar mRNAs can't be resolved.
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Why might mRNA transcripts be similar with different protein products?
1. Paralogs.
2. Splice variants.
3. RNA editing.
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What are paralogs?
When duplications occur, 2 copies of a gene are produced. The 2 begin to change as mutations result in slightly different functions. They are paralogs, e.g. human myoglobin and beta globin genes.
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What is alternative splicing?
Pre-mRNA may be processed differently into mature mRNA by alternative splicing. Splicing removes the introns, and may reconnect the exons in different ways. 40-60% of human genes have splice variants.
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How is there extreme alternative splicing in the drosophila DSCAM gene?
There are 4 sets of alternative exons, which may be connected in any combination. Potentially 38016 mRNA variants.
Human DSCAM has only 3 splice variants.
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What is RNA editing?
Some cells can alter mature mRNA before translation by substitution editing - chemical alteration of specific nucleotides (as in point mutations).
May be:
C -> U (cytidine deaminase).
A -> I (inosine, read as G, adenosine deaminase).
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How does RNA editing alter the human ApoB gene?
ApoB is involved in lipid transport. In the liver there is no editing, resulting in cholesterol transport.
In the intestine, CAA (glutamine) -> UAA (stop), forming a shorter protein for transporting lipids across the intestine wall.
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What is RNAseq?
Sequencing the whole transcriptome (all the mRNA in a cell).
Can detect changes in single bases. Unbiased - changes in expression can be detected in unexpected genes which might not be included in a microarray (useful for cancers caused by rare mutations).
Gene expression can be quantified by read number, more accurate than amount of fluorescence. Can also detect lower levels of expression than microarrays.
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What are the steps of RNAseq?
mRNA is extracted from the cell and converted to cDNA, which is sequenced using NGS.
The reads are aligned to the genome to determine which genes they represent.
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What is an example of using RNAseq?
Psoriasis - RNAseq found 2629 differentially expressed genes while microarrays found 569, however RNAseq missed 46% of the microarray genes - statistical analysis.
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What are microsatellites?
Loci that have many alleles.
In non-coding DNA.
Consists of repeats of a base pair unit (up to 13bps long, usually 2-5).
Usually 10-30 repeats.
Millions of microsatellite loci in eukaryotic genomes.
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Why is there a lot of variation at microsatellite loci?
1. High mutation rate - repeats prone to slipped strand mispairing.
2. In non-coding DNA, so no effect on phenotype.
219
What is slipped strand mispairing?
The new strand and template dissociate and reanneal to the wrong repeat unit in the sequence - the new strand may have one more (loops in the synthesised strand) or one less (loops in template strand) repeat.
220
What are null alleles?
Occur when there is a mutation in the primer target region that prevents amplification.
221
How are microsatellite alleles assayed?
1. Choose loci and design primers.
2. Amplification - PCR.
3. Electrophoresis - gel or capillary.
Multiplex assay:
Several loci run together.
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What are applications of microsatellites?
Identifying individuals:
1. Forensic DNA profiling -amplify and compare sample and suspect microsatellites or compare sample to national DNA database.
Many loci used to be confident of the match (match at 1 locus could be random chance, individuals not unique at 1 locus).
2. Wildlife forensics - illegal whale hunting.
Establishing relatedness:
1. Paternity testing - at each locus, one allele comes from the mother and one from the father. Can determine paternity using mother's genotype and child's. Several loci used to be confident of match.
2. Cooperation - eg. do helper wasps gain indirect fitness by helping? Are they related to breeding wasp?
Identifying population of origin:
1. Assignment tests - where an individual is most likely from, based on knowledge of allele frequencies at microsatellite loci in different populations.
2. Identifying source populations - eg. where did confiscated ivory shipment come from? One population or several?
Determine microsatellite allele frequencies of different populations and the sample to find best match.
*finds probabilities - cannot tell for certain.
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What is the probability of a random match at a locus?
2 x (frequency of allele 1) x (frequency of allele 2).
224
Why might it be difficult to identify species?
1. Don't have the expertise.
2. Don't have the whole organism.
3. 2 different species might look identical.
4. 2 morphs might be the same species.
5. A bacterial species cannot be cultured.
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What is the biological species concept?
Species are groups of actually or potentially interbreeding individuals that can mate and produce fertile offspring.
Species are reproductively isolated.
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Why is mtDNA good for species level identification?
It evolves quickly due to poor proofreading and possibly also the byproducts of cellular respiration, but more slowly than microsatellites, so individuals of the same species are unlikely to differ.
Evolutionary changes are neutral (mutations that don't affect phenotype remain in genome). There can be positive selection but not generally.
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Which mtDNA regions are most commonly used for species ID?
1. Cytochrome b.
2. Cytochrome oxidase 1 (cox1) - barcode gene.
Both coding and quite variable.
Control region (d-loop) can't be used because it is too variable (non-coding).
228
Applications of species ID using mtDNA?
Identify illegally sourced samples:
1. Whale meat sushi - which species? Can identify illegal hunting/international trade.
2. Which badger species does hair come from? Are brush manufacturers using hair from protected badgers? Eurasian badgers protected (with exceptions) but Hog badgers not. Hair mtDNA sequenced for brushes from different companies, cyt b used to determine species and control region to estimate population.
229
Why is cox1 a good barcode gene for animals and what are the problems?
1. Essential for cellular respiration - required by all aerobic organisms.
2. A short sequence with high variability between species and low variability within.
3. Usually high copy numbers (many mitochondria in a cell).
* can't be used for plants because it doesn't evolve quickly enough like in animals. Also structurally diverse.
* gene copies (pseudogenes) can also be transferred to nucleus during evolution, would also be amplified, which would confuse ID.
* prokaryotes don't have mitochondria.
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Applications of cox1 barcode?
1. Species ID - monitor populations of species otherwise hard to identify.
2. Identify prey - to understand predator/prey relationships, food webs, parasitism, interspecific competition.
231
What DNA can be used instead of cox1 to identify plants?
Plastid DNA.
Also maternally inherited and rarely recombine.
No single region has the same discriminatory power for plants as cox1 for animals, but combinations of regions can be used.
For degraded samples, P6 loop of trnL intron used because it is short.
232
Applications of plant species ID?
Natural health products:
Monitor the alternative medicine market by determining product origins.
Important because counterfeit/adulterated products are fraudulent and a health threat.
DNA barcoding can authenticate products.
Surveying fauna - conservation.
Discovery of new species.
Pollen barcoding - how species move.
Ecological forensics - eg niche partitioning.
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Why is below species level identification important?
Can identify harmful strains of bacteria - hyper-virulent lineages.
Can establish source of an outbreak/epidemic.
234
How are bacterial strains ID'd?
No single region varies between but not within strains, so a combination of markers is used.
Housekeeping genes are used because they are expressed in every cell and there are many alleles for housekeeping loci, with each strain having a different one.
Some different strains may have the same allele though, so many loci used.
235
What is multilocus sequencing typing (MLST)?
Uses 7-10 housekeeping loci.
At each locus, each allele is assigned a number.
The probability of 2 strains having the same profile for all loci is very low.
236
What is DNA sequencing?
Determining the order of nucleotides in a DNA molecule.
237
What makes up the genome?
Coding regions.
Non-coding regions.
DNA modifications (epigenetics -methylation, acetylation).
238
What are small and large scale sequencing?
```
Small:
A single DNA fragment.
1 or a few genes.
Target regions inside a gene.
Large:
Whole genome (uses gDNA).
Whole exome (the transcriptome - uses cDNA synthesised from mRNA).
Whole genome bisulfite (methylated regions).
```
239
What brought down the cost of genome sequencing?
Next generation sequencing (rather than Sanger sequencing 1995-2003).
240
What is Sanger sequencing?
Chain termination method.
Requires:
DNA polymerase.
Primers.
Fluorescently labelled ddNTPs.
Produces reads as chromatograms by capillary electrophoresis.
Sequenced human genome in 2003 (took 13 years).
241
What are the advantages and disadvantages of Sanger sequencing?
Advantages:
Reads are easy to interpret.
Reliable.
Disadvantages:
Small scale (samples sequenced individually).
Slow (DNA must be amplified and run on capillary gel).
Expensive + effort.
242
What is second generation sequencing?
Massive parallel sequencing.
| Millions of molecules in parallel, sequencing while synthesising (PCR based).
243
What is next generation sequencing?
Second and third generation.
Advanced sequencing technology.
Software based data analysis and interpretation - assigning bases to dNTP peaks, reassembling sequences (contigs).
High coverage (WGS), depth (no. of reads at one point), throughput (load of data), speed and can compare many samples at the same time.
244
What is third generation sequencing?
Sequencing from a single DNA molecule (not PCR based).
245
How are the sequences assembled?
Reads -> contigs.
| Contigs -> genome.
246
When are Sanger sequencing and NGS used?
```
Sanger:
Single fragment sequencing.
Studies with less than 100 genes.
Bacterial ID/barcoding.
Confirming NGS.
NGS:
Studies with more than 100 genes.
Clinical settings - fast diagnosis.
With limited starting material.
Meta studies.
```
247
When are WGS and WES used?
WGS:
High resolution and coverage.
Identifies potential disease causing mutations in all regions.
Identifies chromosomal rearrangements, deletions and insertions.
WES:
Fine gene expression analysis.
Identifies mutations/polymorphisms in coding regions.
Finds new isoforms or gene variants.
Faster + less expensive than WGS.
248
Potential applications of NGS?
Medicine:
1. Personalised medicine.
2. Genetic diagnosis (before symptoms).
3. Identify known mutations in parents (carrier screening).
4. Prenatal screening.
5. Investigate cause (mutation) of a disease.
Also:
1. Metagenomics - eg. study microbiome, environmental studies.
2. Study gene-environment interaction.
3. Commercial - insurance.
4. Forensics - identification.
5. Individual curiosity (can pay to have own genome sequenced).
249
How are people's genomes diverse?
1. SNPs.
2. Copy number variations.
3. Short deletions/insertions.
4. Chromosomal rearrangements.
5. Microsatellites.
250
Challenges of NGS for medicine?
1. Most variants have unknown medical significance.
2. May find a deleterious/unclear mutation when screening for something else.
3. Right not to know vs. medical obligation to inform.
4. How will data be protected/stored? - insurance, research, possibility of new therapies.
251
When is NGS used in diagnosis?
For diseases that have similar symptoms but different causes.
Pros must outweigh cons.
252
Personalised medicine example?
Warfarin.
Patients with gene variants metabolise the drug differently - polymorphisms affect pharmacodynamics and pharmacokinetics.
253
Why is NGS useful in sequencing microorganisms?
Don't need to be cultured in lab.
Can run many samples at once, allowing comparison.
Useful in studying ecosystems.
Majority of uncultivated microbial taxa can be identified using 16s rRNA.
254
What is human molecular genetics?
1. Locate and characterise genes.
2. Understand the molecular basis for the pathology of inherited disorders.
3. Basis for development of gene based therapy.
255
What are the types of point mutation?
1. Nonsense - results in stop codon or premature termination codon - nonsense mediated decay.
2. Missense - results in different amino acid, can make protein non-functional (depending on properties of AA substituted and location - effect on structure). Rarely beneficial.
3. Silent - no change in amino acid sequence (may still result in altered protein if SNP affects translation kinetics).
256
Types of mutation and diseases?
Point (missense) - sickle cell anaemia.
Deletion - cystic fibrosis (CFTR gene).
Duplication - Huntington's (more repeats).
