Molecular Genetics Flashcards
What is DNA made from?
Deoxyribonucleotides.
Which direction is DNA synthesised in?
5’ to 3’.
How are DNA strands orientated?
Antiparallel.
DNA melting curve - why does fluorescence decrease as temperature increases?
Because intercalating dyes (ethidium bromide, SybrGreen) only bind double stranded DNA.
What is the melting temp. (Tm)?
The point at which there is 50% dsDNA and 50% ssDNA.
How do GC rich sequences affect Tm and why?
They increase it because there are more triple H bonds.
How do mismatches affect Tm and why?
They decrease it because mismatches are unstable and the DNA is therefore more easily denatured.
What does Tm indicate?
The energy required to denature DNA.
What does Tm depend on (3)?
- Proportion of A-T:G-C.
- Mismatching.
- Fragment length.
How is Tm used?
To identify DNA variations: Alleles. Polymorphisms. Mutations. Contaminations/infections.
How was cloning done before PCR?
Using cloning vectors with DNA fragments inserted and introducing them to host cells to grow into colonies.
Why is PCR better than using cloning vectors?
It is quicker and easier, and cloning vectors often would not work.
How does PCR work?
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).
What are the PCR reagents (5)?
- Template DNA.
- Primers.
- dNTPs.
- Buffer.
- DNA polymerase enzyme.
What technology is used to carry out PCR?
A thermo-cycler.
What do primers provide in PCR?
Specificity and directionality.
What are the three stages of PCR?
- Denaturation.
- Annealing.
- Extension.
What is electrophoresis used for?
PCR visualisation.
What is used as a conductor in electrophoresis?
Ionic buffer.
Which direction does DNA move in during electrophoresis?
Towards the positive electrode.
How are PCR products visualised in electrophoresis?
Using intercalating fluorescent dyes.
Why are reaction efficiency and accuracy lower when there are more cycles?
The reagents begin to run out as the amplification curve approaches the plateau. At this point, PCR is prone to errors.
When is PCR amplification exponential?
When there is an abundance of reagents.
What is an application of end point PCR?
Detection of size polymorphisms (fragments are separated by size in electrophoresis).
What are the benefits of end point PCR?
- Sensitive (only 1 DNA molecule needed).
- Specific (primers).
- Fast.
- Flexible (adapted for different investigative purposes).
What is the downside of end point PCR?
It is not quantitative.
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.
How is qPCR used?
- Gene expression.
- Detecting bacterial contamination eg. of water.
- Detecting food contamination (e.g. horse meat)/GMO.
- Detecting contamination of crime scene DNA.
- Human diagnostics/genetic screening.
- Detecting infection/cancer progression.
How is rtPCR DNA visualised?
- Intercalating dyes.
2. Fluorophore-linked oligonucleotide TaqMAN probe.
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.
How do TaqMAN probes work?
They contain a fluorescent dye and a quencher. When the 2 are separated by hydrolysis, light is visible.
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.
What are the advantages of using intercalating dyes over TaqMAN probes?
They are easier and cheaper.
Where are TaqMAN probes most used?
In clinical and environmental diagnostics.
How is fluorescence detected in rtPCR?
Using a photodetector.
What is fluorescence proportional to in rtPCR?
Amplified DNA.
What are the 2 main parameters given by qPCR?
- Visualisation of amplification curves for quantification (accumulation of fluorescent signals gives amount).
- DNA melting curve for product identification.
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.
When there is more infection/contamination, what happens to the Ct value?
It shifts to the left.
What is analytical sensitivity?
How often a positive result is returned when the target is present.
What is analytical specificity?
How often a negative result is returned in the absence of the target.
In what areas does PCR have important applications?
- Medicine.
- Environment.
- Forensics.
- Evolution.
- Research.
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).
What is COLD PCR?
Co-amplification at lower denaturation temperature PCR.
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%).
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.
When is cDNA used?
For amplification when from starting from mRNA or virus RNA.
How is cDNA made?
Retrotranscriptase. A linear reaction.
Why is specificity of rtPCR important?
Better diagnosis and treatment - different drugs for different bacterial/viral strains.
What is serology?
Blood test to detect antibodies in the patient (measure immune response). Can be compared to pathogen (genome detected by rtPCR).
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.
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.
What are 4 mosquito borne viruses?
- Dengue (4 serotypes).
- West Nile.
- Chikungunya.
- Zika.
Where is Dengue found?
Countries in C/S America, Africa, Asia. Subtropical and highly populated but spreading further North into Europe (warming).
What are symptoms of dengue fever (febrile phase)?
Sudden onset fever, headache, nose/mouth bleeding, muscle/joint pain, vomiting and diarrhoea, rash.
What are symptoms of dengue fever critical phase (haemorrhagic fever)?
Hypotension, pleural effusion, ascites, GI bleeding.
What are symptoms of dengue fever recovery phase?
Altered consciousness, seizures, itching, slow heart rate.
How is rtPCR applied to dengue?
Early detection and intervention. Preventing epidemics. Identifying the strains to decide the best treatment and identify new mutations.
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.
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).
What are the stages of HIV infection?
- Acute - high viraemia, flu like symptoms.
- Asymptomatic but progressive.
- AIDS - increasing viraemia, infections and proliferative disorders.
When is qPCR used for HIV patients?
- At entry into care.
- Every 3-6 months in untreated patients to monitor progression.
- 2-8 weeks after starting HAART drug therapy.
- Every 4-8 weeks until the viral load is below detectable limits.
- 2-8 weeks after changing therapy.
- Every 6-12 months to monitor effectiveness of therapy.
What are parameters of HIV infection?
- CD4+ levels.
2. Viral load (viraemia).
What CD4+ levels show in HIV?
Decreasing CD4+ = worsening infection.
Increasing CD4+ = improving infection.
What are the symptoms of Ebola?
Days 7-9 = headache, fatigue, fever, muscle soreness.
Day 10 = sudden high fever, vomiting blood, passive behaviour.
How are IgM and IgG antibodies measured?
ELISA.
When does viraemia peak for Ebola?
With the onset of symptoms (day 7). Viraemia is a strong predictor of outcome.
How long after infection are IgM and IgG detected?
IgM = 3-6 months. IgM = 3-5+ years (possibly lifelong).
When in the course of the disease is qPCR used?
Within days of onset of symptoms.
Why is analytical sensitivity critical for Ebola?
Early detection is important in preventing spread.
Patient discharge is based on a -ve qPCR result.
What is nitrification?
Ammonium -> nitrite (nitrosomonas) -> nitrate (nitrobacter).
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).
Issues in food security:
Need enough food to meet human demand, produced sustainably.
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.
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.
What is produced from oil refining?
Household goods - lubricants, waxes, polishes.
Petrochemical products - pharmaceuticals, industrial/specialty chemicals.
Asphalt.
Fuel.
Based on existing reserves, when will fossil fuels run out?
Oil - 50yrs.
Gas - 60yrs.
Coal - 120yrs.
How much of oil consumed globally is from fossil reserves as opposed to renewable resources?
97%.
How are biofuels and chemicals produced?
Fermentation of plant sugars.
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.
What are green revolution initiatives?
Increased agricultural production: High yielding crop varieties. Fertilisers. Irrigation. New (mechanised) cultivation methods. =Tripled production and yield.
Issues for a second green revolution:
- Restricted land use.
- Competition with non-food agriculture.
- Quality must be improved.
- Chemical use and input resources (e.g. water) must be reduced.
- Must be tolerant to erratic environmental conditions (climate change).
What are sources of genetic variation for crop improvement?
- Genetic collections.
- Wild populations.
- Induced mutations.
- Breeding.
- Genetic manipulation.
May be closely related (primary), more distantly related (secondary), marginally related (tertiary), not at all related (quaternary - all organisms, transgenic by GM).
What is conventional breeding?
Selectively breeding for better traits. Uses natural variation. Has resulted in crops very different from their ancestors.
What is marker assisted selection?
Using molecular markers to assist selective breeding.
What are genebanks?
Maintain diversity from natural variation in tissue culture.
What is mutation breeding?
Creating random mutations using radiation or chemical mutagens. First released variety improved tobacco quality (Indonesia, 1934).
What are commonly used chemical mutagens?
- Alkylating agents - EMS, MNU…
- Sodium azide.
Also other chemicals.
Plant material is soaked in specific conc. for specific length of time.
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.
What types of mutations can chemical and physical mutagens cause?
Chemical: Point mutations. Insertions and deletions. Physical: Above. Duplications. Translocations. Inversions.
Are more mutant crops produced by physical or chemical mutagens?
Physical.
What is an example of a mutant crop?
Calrose 76 semi-dwarf rice produced by gamma irradiation resulted in a 15% yield increase.
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.
What does GM do?
Produces transgenic plants. Foreign genes are introduced and expressed in plant cells to give the plant a new, useful characteristic.
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.
When we’re GM plants first created?
Early 1980s.
What were the first plants genetically modified?
Tobacco, petunia and sunflower.
What are the steps of genetic modification?
- Insertion of gene of interest into vector (T1 plasmid - A. tumefaciens, restriction and ligation).
- Delivery of the vector into plant cells.
- Moment of the DNA to the nucleus and integration into the genome.
- Selection of transgenic cells.
- Regeneration of those cells into transgenic plants.
- Passed to future generations by Mendelian inheritance.
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.
What does T-DNA of the Ti plasmid contain?
Oncogenic genes - auxin and cytokinin biosynthesis. Overproduction produces callus - crown gall disease
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.
How are whole transgenic plants grown from the transformed cells?
They are grown on media with auxin and cytokinin.