Diagnostic Technologies

Question 0

Types of FISH

Answer 0

Repeat sequence: probes isolated from telomere/centromere regions to detect gain or loss of chromosomes
Single copy: probe isolated from cloned DNA of a disease causing gene or a fragment of DNA of known location associated with particular gene, to detect gene, gene region or chromosomal rearrangement
Subtelomere: from distal ends of chromosomes in regions proximal to actual telomere regions, gene rich, identify very small deletions/rearrangements
Chromosome painting: cocktail of many DNA fragments, entire chromosome fluoresces, identify complex rearrangements

Question 1

FISH

Answer 1

To determine if gene, mutation, or chromosome rearrangement is present/absent
Uses molecular probes (from known fragment of DNA labeled with fluorescent dye) to hybridize to chromosomes instead of dyes that bind DNA or protein of chromosome
Use internal control in different region of same chromosome to rule out failure of probe to bind
No signal at site of disease location means true deletion
Locus specific and chromosome specific
Probes covers just a critical region not entire deletion so deletion may be present but not detected

Question 2

Contiguous gene syndromes

Answer 2

Regions in genome with clusters of closely associated genes whose normal functions are generally unrelated
Deletions and duplication results in multiple phenotypic anomalies (syndrome)

Question 3

Williams syndrome

Answer 3

Deletion of elastin gene (ELN)

Coarse hair, skin, lack of flexibility in aorta, developmental problems, excellent musical skills

Question 4

Velocardiofacial syndrome

Answer 4

Diagnoses in infancy: difficulty feeding, cardiac defects, facial dysmorphologies
Learning disabilities, short stature, conductive hearing loss
Deletions due to unequal crossing over
Generally passed on, alleles on normal chromosome may compensate for missing deleted chromosome in parent but child will get different chromosome from other parent that can’t compensate

Question 5

Microarray

Answer 5

Test DNA and reference DNA labeled with different colors and binds to probes (evolved from southern blotting)
DNA (gene) arrays: identify genetic polymorphisms, specific mutations, copy number variation (CNV)
Expression arrays: DNA fragments representative of genome on slide, cDNA labeled and hybridizes, high expression is red, low expression is green
Chromosome microarray: view CNV in association with chromosome, hybridization is same as in gene array but data here are plotted in order along lengths of each chromosome

Question 6

Next generation sequencing

Answer 6

Genome wise scan to detect mutations

Targeted sequencing to simplify and maximize gains

Question 7

Compare: 
Karyotype 
Molecular diagnostics 
FISH
Microarray
DNA sequencing

Answer 7

Karyotype: relatively large, numerical/structural abnormalities, genome wide
Molecular diagnostics: well defined, specific, very small mutations, targeted testing
FISH: well defined, specific, medium mutations, targeted testing
Microarray: generalized genome wide screen for small to large mutations, will not detect balanced rearrangements
DNA sequencing: high resolution for targeted regions to detect mutations to single base level