Genetic Variation and Disease #1 Flashcards
What is an allele
An alternate form of a genetic variant (must be located at the same place in a gene)
What does having genetic variants for a gene mean?
You could have two different versions of a protein
Describe the the reasons why we can track the inheritance of genetic variants
- When a genetic variant causes a visible/measurable change in phenotype we can track its inheritance pattern
- An individuals phenotype depends on their genotype
- Genotype: combination of alleles a person has for a particular genetic variant
- The simplest forms of inheritance to tract are dominant and recessive traits
Describe loss of function and gain of function
- The mode of inheritance an allele has depends on what the allele does to the protein and how the protein functions
Gain of function:
- A gain of function allele causes the protein to weak too well or do something new
- often show a dominant inheritance pattern (because its is hard to ‘hide’ the gain in function. ie. protein from another allele can’t compensate for the new function)
Loss of function:
- a loss of function allele causes the protein to no longer work
- often show a recessive inheritance pattern (because usually the other allele producing a functioning protein can compensate for the lost function)
- however this will not be the case if oaring only one functioning allele does not produce enough protein
Monogenic traits vs. polygenic traits
Monogenic:
An inherited characteristic that is controlled by a single gene
- Discrete traits (have or don’t have)
Usually follows simple recessive/dominant inheritance patterns
- can track who will have the trait/condition based on their genotype
Environment may influence exact presentation of trait
Polygenic:
An inherited trait/charactaristsic that is controlled by multiple genes
- includes traits on a continuous scale such as height
Do not follow an obvious inheritance pattern. ie:
- common in a family but can the categorised into dominant and recessive
- often talked about in terms of likelihood and risks
Also usually influenced by environment
Describe Retinis Pigmentosa (gain of function variant) (memorise key concepts)
Retinal degenerative disease caused by variants in Rhodopsin receptor protein
- Symptoms: night-blindness, loss of complete vision over time
- Dominant inheritance pattern
Rhodopsin receptor located in rod cells in eyes (detects presence of light and sends signals to brain, which are turned into an image)
(GPCR) Receptor has an ‘ion lock’ to keep completely inactive when no light present (even tho ligand is bound all the time)
- Helps make receptors extremely responsive to light
- genetic variants that interrupt this ‘ionic lock’ make rhodopsin constantly active
- leads to death of rod cells and loss of sight over time
Describe Phenylketonuria (PKU) - loss of function variant (understand key concepts, not necessarily memorise specific stuff)
Inherited inability to metabolise phenylalanine
- symptoms: if untreated, causes brain damage and nerve damage
- recessive inheritance pattern
Phenylalanine hydroxylate converts phenylalanine to tyrosine (which is then broken down further)
>950 different variants recorded causing loss of enzyme function
- leads to accumulation of phenylalanine and decrease in tyrosine
- increased phenylalanine in brain leads to brain damage
Can be treated with a low-phenylalanine diet to ensure no build-up occurs and avoid severe symptoms
Describe genotyping
The process of determining the specific genetic alleles in an organisms DNA
- many diff methods (PCR-RFLP, fluorescence-based methods, genome sequencing) choose method based on what you want to look at
Often want to confirm presence or absense of disease causing allele.
- could help point to specific causes of disease
- confirm disease inheritance pattern
- could produce future health/disease progression
Describe the components and steps of PCR (polymerase chain reaction)
lab based method to make many copies of specific segment of DNA
Components:
- DNA sample: DNA extracted from organism’s cells
- Primers: small segment of DNA complementary to specific sequence surrounding DNA of interest
- Polymerase: heat-tolerant DNA polymerase enzyme (not from our cells)
- Nucleotides (dNTPs): DNA nucleotides to build new DNA strands with
Steps:
- Denaturation: DNA is separated by heating to a very high temperature
- Annealing: reaction is cooled so DNA primers can attache to target sequence
- Elongation: heat increased slightly so heat tolerant DNA polymerase can bind primer and replicate DNA
Exponential growth, usually only need 25-30 cycles
Describe the PCR-RFLP method of genotyping
Polymerase Chain Reaction-Restriction Fragment Length Polymerisation
- Genotyping that uses PCR, restriction enzymes and gel electrophoresis to determine genotype
- quick and simple. easy to run in any DNA lab. scalable (can get a few samples at once up to ~400 per gel)
What is a restriction enzyme
Restriction enzymes are proteins isolated from bacteria that cut DNA
- a bacterial defence mechanism
Each restriction enzyme recognises a particular sequence and cuts that sequence at a particular base
- recognised sequence is short (4-6 bases) and palindromic
- enzyme cuts the same place on both DNA strands
10s of restriction enzymes exist, so 100s of possible recognition sequences
Describe the process of using the PCR-RFLP method
- PCR primers are designed to amplify DA surrounding variant of interest
- Restriction enzyme is chosen based on ability to cut one allele of the variant but not the other allele
- DNA fragments (after PCR and restriction digest) are separated by size on DNA gel
- smaller fragments travel further through gel
- cut DNA has two fragments, uncut DNA has one fragment
Results:
- no cut and one strand seen means no variation
- two cuts and two strands seen means both strands had the variation
- one cut and three strands seen means one strand had the variation
Describe the fluorescence method of genotyping
Fluorescence based genotyping methods use different ‘coloured’ probes to detect alleles
Process:
- PCR primers are designed to amplify DNA surrounding genetic variant of interest
- Probes are designed to specifically anneal to one of the two alleles
- each probe has a different fluorescent dye attached
- as polymerase amplifies the DNA the probe is broken down and releases the dye
- the specialised PCR machine (called a LightCycler) detects the colour of the dye upon release
What results do fluorescence methods give
Clustering graphs calculate which alleles are present in a sample genotypes using fluorescence methods
- requires enough samples to form a cluster, not very useful if only genotyping one or two samples
Genotyping ‘chips’ use flueorscence based methods to assess multiple genetic variants at once (50,000-100,000 at once)
Homozygote colours will be at either corners of the graph and the colour in the middle represents the heterozygotes (as the colours of each probe have blended to make the middle colour seen).
