Medgen exam Flashcards
Define gene therapy
The introduction of a healthy gene in order to replace an absent or mutated gene to correct a genetic disorder
Outline DNA extraction procedure
- Isolate cells
- Add lysis buffer: contains detergent which lyses cells to release DNA, also contains proteinase K which breaks down histones
- Add salt solution: proteins + debris clump together
- spin down to pellet debris + proteins
- Add isopropyl alcahol to precipitate out DNA
- spin down + remove supernatant- you now have a pellet of DNA- add buffer to redilute
Outline Taqman assay
In the reaction vessel you will have normal PCR reagents including Ampli Taq Gold DNA polymerase as well as two Taqman probes that are correspond to the two possible SNPs you are genotyping.
The probes have either a FAM or VIC dye on the 5’ end which flouresce at two different wavelengths as well as a minor groove binder and none flourescent quencher on the 3’ end. The quencher suppresses any flourescence fro the VIC or FAM dye.
After the denaturation step the complementary probe will anneal to the ssDNA between where the forward and reverse primers anneal.
When Taq DNA polymerase extends from the primers and reaches the probe the taq exonuclease cleaves the probe bound to the DNA releasing the dye from the quencher.
The dye then fluoresces corresponding to the probe that bound, this can be quantified by RT-PCR
Outline sexting PCR
Do a normal PCR with primers for both the X and Y chromosome amelogenin gene on each chromosome the amelogenin gene is two different sizes, run the products on a gel- females have 1 band, males have 2
What is linkage disequilibrium?
The none random association of alleles at two different loci
For a population with three haplotypes, what can you infer about D’?
In this case D’ = 1, this is referred to as Complete LD, the two loci are not being separated by a recombination in this population since at least one of the haplotypes does not occur in the population
For a population with two haplotypes, what can you infer about r2?
In this case r2 = 1, it is referred to as Perfect LD. Perfect LD occurs when there are exactly 2 of the 4 possible haplotypes present in the population, and as a result, the two loci also have the same allele frequencies.
Loci that are in perfect LD are not necessarily in complete LD Linkage Disequilibrium D’ and r2- if the two loci both have very rare alleles and the rare alleles do not occur together on a haplotype, it is possible for D’ to be 1 (since 1 of the haplotypes does not occur in the populations) and for r2 to be small (when the alleles at the two loci for the 3 remaining haplotypes are not correlated).
Why might there be a difference between the expected and observed allele freq?
Small sample size
Selection
genotyping error- one of your probes doesn’t bind to one of the alleles as effectively
new mutation
Describe what an enhancer is and its mode of action
Cis acting regulatory DNA sequence that can work in convert with activator proteins to increase transcription.
- Activator protein binds enhancer region
- DNA looping
- Activator proteins interact with the transcription initiation complex via various cofactors
- Transcription is activated
Enhancers can also recruit RNA polymerase and general transcription factors to the promotor
Describe what a silencer is and its mode of action
Cis acting regulatory DNA sequences that work in concert with repressor proteins to inhibit transcription
- Repressor protein binds to silencer
- Triggers DNA looping
- Repressor proteins bind to specific transcription factor binding sites the assembly of the transcription initiation machinery
Define an insulator
DNA element that inhibits transcription by preventing heterochromatin spreading or action of cis acting enhancers.
Whole-genome sequencing analysis identifies a synonymous variant in an individual with early-onset diabetes and a strong family history of diabetes. What further information would help to determine the likely pathogenicity of this variant?
Determining whether the variant segregates with disease in the family (1)
Do other mutations in this gene cause early-onset diabetes (1)
Frequency of this variant in individuals without early-onset diabetes (1)
Has a mutation in the same gene/different gene, and known to cause early-onset diabetes, been found in this individual? (1)
Has this variant been seen in other individuals with early-onset diabetes and without a genetic diagnosis? (1)
When might you suspect LD?
When the population frequency of a haplotype is significantly different from the frequencies of the individual alleles. I.e. if alleles were fairly rare in the population but that specific combination had a very high population frequency
Why does the number of specific mutations vary in different tumours?
Melanomas and lung tumours display many more mutations than other tumours because the skin and lungs are exposed to the highest levels of carginogens
Paediatric tumours display fewer point mutations because the number of point mutations increases with age
Cancers which involve mutation of DNA repair genes, eg Lynch syndrome may have many more mutations present than tumours where these genes are not mutated
Describe a viral gene therapy
Luxterna- gene therapy developed to treat cases of Leber congenital amaurosis caused by the RPE65 mutation, LCA- hereditary condition that affects vision, symptoms develop in the first few months of life, sufferers will eventually go blind, LCA is caused by mutations in various genes. Luxterna uses an AAV2 vector virus to deliver heathy RPE65 DNA to the retina cells, it is administered via subretinal injection and has been shown to be effective at reducing progression in children, nhs has declared they will fund luxterna in September 2019
