Molecular Testing Flashcards
(25 cards)
Germline Mutation
Inhereted mutations present in every cell
Somatic Mutation
Acquired mutations present in diseased tissue
Clinical Utility of molecular diagnostic tests for germline mutations
Confirms diagnosis
Screen at risk mutation carriers
Prenatal diagnosis
Screening populations
Pharmacogenetic testing (how genes dictate response to drugs)
Clinical Utility of molecular diagnostic tests for somatic mutations
Identification of tumours and prediction of tumour response to chemotherapy
Prognosis
Challenge facing molecular tests
Huge variety of mutations that require various different tests
Mutation hotspots example
Mutations in Hungtington’s disease occur in polyQ region of HTT gene
In CF, 80% mutations are in ΔF508
They are usually in functionally important areas
Duchenne’s and Becker’s Muscular Dystrophy
X-linked recessive Deletions in dystrophin gene
Different mutations in same gene cause different phenotype
DMD due to frameshift mutations cuasing truncated protein and total loss of function
BMD due to in-frame deletions causing partial loss of function
60% of mutations in both occur in two hot spots
How does the dystrophin protein work
A huge membrane bound protein that helps with calcium release and contraction of actin fibres
Describe symptom development of DMD and BMD
DMD - symptoms appear 2-5 yo; loss of ambulation by 12. Death by cardiac/respiratory complications in 3rd decade
BMD - May survive to old age
How does locus Heterogeneity affect molecular testing
Increases the amount of testing needed as one symptom may arise from the dysfunction of many different proteins (e.g. one protein in a complex)
Imprinting
Only one allele is expressed from birth; silencing done by epigenetic modification
Deletion of a gene whether on maternal or paternal gene affects syndrome
Where is DNA used in genetic tests derived from
Lymphocytes in blood mainly
Sometimes mouthwash cell/buccal scrapes from babies
Chorionic villi/amniocentesis
Why is PCR such a game changer
Requires tiny quantities of starting material to produce huge amounts of target product which can then be analysed using various assays
Expansion mutation
Form of mutation involving expansion of triplet repeat sequences in coding or non-coding region
Anticipation characteristic of expansion mutations
Age of onset is lower and/or worse and/or more common in successive generations as progressive repeats makes mutation worse
Different diseases have different thresholds as this leaves out any residual function
(Huntington’s has >40)
How can polymorphic mutations ease post PCR analysis
Can be used as markers even for when mutations are unknown (generally near locus of mutated gene)
Multiplex PCR
Using several primers to amplify several regions simultaneously to then be analysed
Figure out how to identify this and what this tells you… It’s not that hard you did it in school
figure out Multiplex Ligation Probe
Methylation specific PCR
When DNA is methylated; they are modified by bisulphite reaction where methylated cytosines are resistant to bisulfites but non-methylated get converted to cytosine
Methylation specific primers can be used to test for presence/absence of PCR product
We know which allele on which parental chromosome should be methylated in what way, so the results from analysis can indicate where mutation is and what the syndrome is (where revelant)
Mutation Scanning
If we have multiple PCR products but know the mutation is only in one place, we can narrow down the analysis by using the physical characteristics of the strand like heteroduplex formation or other methods
For analysis of PCR products, what is the most definitive answer
Sequencing
Methods of sequencing
Dideoxy/Sanger sequencing
Pyroseqencing
Compare pyrosequencing and sanger sequencing
Pyrosequencing is more sensitive but cannot sequence as much length of DNA as sanger
