Lecture 5 Flashcards
what is electrophoresis
separating DNA molecules according to their size
To see bands of DNA on a gel, stain with Ethidium Bromide and expose the gel to UV light. DNA fluoresces purple.
Note that Ethidium Bromide binds directly to the DNA.
If Ethidium Bromide binds to your DNA it will damage it.
So Ethidium Bromide is mutagenic & carcinogenic.
how do you detect STRs
STRs are deleted at single sites. we all have 2 alleles so each person will have 2 copies, but the copies can be different lengths (heterozygous) or the same length (homozygous). STRs are hyper variable. STRs are inherited in mandolin fashion. detection depends on size- STRs are small enough for PCR
Get DNA sample, even a miniscule sample will do
Use PCR amplify across the repeated sequence from unique sites flanking the repeat. Run on an electrophoretic gel and visualise the bands.
In reality there is likely to be a lot of population variation in an STR, but each individual will have two alleles.
how much variation is there in STRs
A single 4bp STR showing 11 to 100 repeats has 90 variants
As we all have two alleles, so variation at this STR is 8100 genotypes Actual variation is less than this because:
Not all STR alleles are present at similar frequencies
Human populations are not randomly mating Note the current total human population is 7 billion!
So Forensic Scientists score several STRs simultaneously
describe how dna profiling uses 13 “markers”
Each marker is very variable Each person has 26 markers
13 from their mother 13 from their father
This is stored as 26 numbers Everyone is different except identical twins
describe hungtingtons disease
progressive brain disorder that causes uncontrolled movements, emotional problems and loss of thinking ability
adult onset huxngtingtons usually occurs in a person’s 30s or 40s
early damage is most prominent in the striatum, but other areas of the brain are effected as the disease progresses
prevalace is 5-10 per 100000 people
describe the mutation in hungtingtons disease
dominant mutation on chromosome 4
has high penetrance which means those who inherit mutant allele will have the disease
there is anticipation which means the disease symptoms tend to get more severe and occur earlier with each successive generation
describe the hunttingtons protein
glutamine repeats cause protein misfiling and aggregation
translation of irregular Htt proteins with expanded polyglutamine domains signals to the cell that these proteins are abnormal, causing the cell to recruit proteases to cut these proteins up into small fragments that can be recycled and digested by the cell
unfortunately, some of these fragments can get into the nucleus of the cell forming aggregates that disrupt the functions of the cell, such as transcription.
the smallest and most harmful Htt protein fragments are caused not by it being cut up by proteases but rather by abnormal alternative splicing of the Htt mRNA, leading ti a very small protein fragment that can enter the nucleus. ultimately can become toxic to the nerve cell.
what are the symptoms of myotonic dystrophy
muscle weakness myotonia abnormal heart rhythm chest and breathing problems digestive problems cataracts problems with thinking and planning
describe myotonic dystrophy
one of the most complex known disorders with huge variance in clinical symptoms.
it has variable penetrance which means there is considerable difference in symptoms between individuals, even in the same family
the genetic defect can be slightly different in various tissues in a single individual and can change over time (somatic mosaicism)
there is anticipation which means the disease symptoms tend to get more severe and occur earlier with each successive generation
transmission is nearly always maternal for the congenital form
describe the myotonic dystrophy phenotype
myotonic dystrophy was the first autosomal dominant disease found to be caused by a repeat expansion that is transcribed into mRNA but is not translated into a protein
the nucleotide repeats cause the RNA strands to develop abnormal hairpin folds. these misshapen RNA structures then bind splice-regulating proteins, forming RNA-protein complexes that accumulate within nuclei
these nuclear foci can disrupt biological function by alternating the available amounts of 2 classes of RNA binding splice regulators
MBLN1 regulators are sequestered in the nuclear foci, resulting in depletion and loss of function
increased levels of other proteins such as CUG triplet repeat binding protein 1 leads to misregulation of global splicing events
the disruption of these splice regulators interferes with the processing of transcripts in more than twenty other genes
describe MBNL1
in MD1 the long strands of RNA stay in the nuclei of cells and protein molecules such as MBNL1 and MBNL2 become stuck to them
loss of MBNL1s normal function leads to improper splicing of several proteins such as some of those involved in muscle contraction and growth
MBNL2 appears to act at the genetic level to help maintain muscle-fibre structure, such as collagens
describe route of HIV infection
CCR5 is a G protein coupled receptor that normally controls migration of white blood cells from the blood to inflamed tissue. G Proteins are a protein family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and cellular responses
CD4 is a receptor on T cells
HIV entry requires CD4 and CCR5
some chemokines, such as RANTES block HI entry
how does variation at the CCR5 receptor cause variation in susceptibility to HIV
Variation at the CCR5 Receptor Gene
The CCR5D32 allele carries a 32bp deletion leading to production of a non-functional CCR5 protein.
CCR5D32 confers HIV resistance.
CCR5D32 few % in North Europe. CCR5D32 not in other populations. CCR5D32 has Viking origin? CCR5D32 spread by EuroEmpires?
Other mutant CCR5 alleles have partial function an partial resistance.
No CCR5: No Infection
how does variation at ligand genes cause variation in susceptibility to HIV
Note Nomenclature Chaos.
CCL3L1 shows strong binding to CCR5 (like RANTES in cartoon)
CCL3L1 down-regulates CCR5 by binding to and internalising it.
Variation in CCL3L1 gene copies from 0 to 11 per chromosome.
Some ethnicity effect: Africans 4 copies Pygmies 10 copies
Chemokine (C-C motif) ligand 3-like 1
More CCL3L1: Less Infection
west nile virus infection:
Will future humans be:
- Null mutant for CCR5 receptor?
- High CCL3L1 ligand copy number?
Probably not in Africa because:
West Nile Virus needs CCR5 receptor function to encourage migration of white blood cells from the blood to inflamed brain tissue. So CCR5 null mutants have lower survival rate. Effect of CCL3L1 ligand copy number on survival is less clear
Note that we only see such profound genotype differences in HIV survival rates because HIV is a new disease.
