click below
click below
Normal Size Small Size show me how
BF - MM5
Testing for Genetic Disease
Question | Answer |
---|---|
Why do genetic testing? | Diagnostic, mutation detection, predictive testing, prenatal testing |
Why is counselling and consent needed? 1. Info can be...? 2. Can indicated individual's...? 3. Can have serious implications for...? 4. Test can give.../...results? | 1. Complex 2. Future health 3. Pregnancy/ children/ relatives 4. Unexpected/ uncertain |
Where can the sample be taken from? | Any tissue with nucleated cells (normally blood) |
Cytogenetic testing - What is added to blood sample first? | Anticoagulant |
Cytogenetic testing - What is purified? | Lymphocytes -> cultured for 3-4 days |
Cytogenetic testing - What needs to be done during mitosis of sample lymphocytes? | Disrupt mitotic spindle to prevent completion |
Cytogenetic testing - The cells are then harvested and treated with what kind of solution? | Hypotonic |
Cytogenetic testing - What is then done with these swelled cells? | Fix to a microscope slide and stain |
Cytogenetic testing - What is then constructed from the images seen down the microscope? | Karyotype |
FISH - What does it stand for? | Fluorescence in situ hybridization |
FISH - What is first constructed? | A DNA 'probe' (copy of target region) |
FISH - How is this DNA 'probe' labelled? | Fluorescently |
FISH - What is the probe applied to? | A target chromosome |
FISH - What two things are done during application of probe to chromosome? | Incubate and hybridize |
FISH - Wash step. What is seen in a normal result? | Probe binds twice to expected chromosomes |
ARRAY-CHG - What does this test allow us to do? | Test thousands/ millions of genomic regions at a time |
ARRAY-CHG - What must be used as a containing to carry out this test? | Inert (glass or metal) welled tray |
ARRAY-CHG - What are fixed to specific wells? | Multiple DNA probes |
ARRAY-CHG - What is added to the well? | Patient DNA sample and reference DNA sample added in equal amounts |
ARRAY-CHG - How is the patient sample and reference sample distinguished? | Different colours |
ARRAY-CHG - How can different coloured wells identify genetic abnormalities? | If reference = test (mixture of two colours), if the colour is predominantly that of reference = deletion in patients DNA, if colour is predominantly that of patients sample = duplication in patients DNA |
DNA Sequencing - How many reactions are set up? | 4 |
DNA Sequencing - What are each of these 4 reactions based on? | A,T,C,G - deoxynucleotides for each base with radio labels |
DNA Sequencing - Why are deoxynucleotides used? | They will terminate any existing DNA sequence they are added to |
DNA Sequencing - What must be first done to test DNA? | PCR -> cleaved into fragments of different lengths |
DNA Sequencing - What other 4 components are added to the reaction? | DNA polymerase, nucleotides (normal), primers, fragemented test DNA |
DNA Sequencing - What is produced? | Different fragments of DNA with a deoxynucleotide base terminating each fragment |
DNA Sequencing - What is then done to these fragments? | Electrophoresis to sort them by length |
DNA Sequencing - Those of the same length...? | Will move the same distance in the gel and have the same terminal base |
DNA Sequencing - How is sequence read from gel? | Autoradiography (x-ray), to detect the radio labelled bases, sequence is then determined |
DNA Sequencing - How are modern techniques different? | Flourescently labelled ddNTP terminators that can be read automatically |
DNA Mutations - What does VUS mean in terms of mutations? | Variance of unknown significance |
DNA Mutations - How can you decide if a mutation is pathogenic, benign or VUS? (4) | 1. Significant protein change (nonsense/ frameshift) 2. Other affected family members 3. Not present in control/ normal population 4. The residue is functionally important (e.g. binding site) |
Why do genetic tests have varying sensitivity? 1. Test rarely covers...? 2. Test may miss...? 3. Disease may be due to...? 4. Mutation may be present in only some...? | 1. All of a gene 2. Certain types of changes 3. Changes in another gene 4. Of patients cells (e.g. mosaicism) |
Extras - DNA testing focuses more on introns/exons/boundries? | Exons and intron/exon boundaries |
Extras - Why is DNA testing focused more on exons? | Thought to contain more pathogenic changes |
Extras - Is DNA testing sensitive to larger deletions? | No |
Extras - What does MLPA stand for? | Multiplex Ligation-dependant Probe Amplification |
Extras - What does MLPA show? | Large deletions or duplications |
Extras - Do negative results from DNA testing exclude a diagnoses? (what is exception) | No - except when testing for specific known familial mutation |
Extras - What can gene tests do to assess probability? e.g. | Some are good at giving negative predictive values e.g. for CF and HD |
Extras - What are the two methods of obtaining prenatal genetic information? | Chrionic Villus Smapling (CVS) and Amniocentesis |
Extras - When and how is CVS done? | 10-12 weeks -> placenta biopsy |
Extras - When and how is amniocentesis done? | 15+ weeks -> amniotic fluid |
Extras - CVS/amniocentesis have greatest risk? | CVS (2%), Amnio (1%) |