Molecular Diagnostics Flashcards
What are some disorders that can be defined by karyotyping?
Down syndrome (trisomy 21)
Edward’s syndrome (trisomy 18)
Turner’s syndrome (monosomy X)
Klinefelter’s syndrome (XXY)
Cri-du-chat (deletion of 5p)
What are some signs of Edward’s syndrome?
Small mouth, jaw and short neck
Shield chest
Occiput
Malformed ears
Clenched hands with overlapping fingers
Flexed big toe
Signs: Short stature
Low hairline
Widely-spaced nipples
Brown spots (nevi)
No menstruation
Constriction of aorta
Rudimentary ovaries
Diagnosis ?
Turner’s syndrome
What are some signs of Klinefelter’s syndrome?
Frontal baldness absent
Fewer chest hair and poor beard growth
Narrow shoulders
Gynecomastia
Wide hips
Small testicular size
Long arms and legs
What are signs of cri-du-chat?
Abnormal development of glottis and larynx; high pitched cries
Microcephaly
Round face
Downward eyes
Small chin
Short neck
What is karyotyping?
A pictorial display of metaphase chromosomes from a mitotic cell
Why are chromosomes in metaphase (karyotyping)?
Chromosomes are more dense during metaphase so easier to view
What is the process of karyotyping ?
Harvest cells are cultured
Cells treated with colchicine, stained to be observed
Chromosomes are photographed
Chromosomes are identified by side, position of centromere and banding and staining regions.
What is amniocentesis?
Obtaining amniotic fluid which has cells from the fetus to examine for any disorders
When is amniocentesis done?
Between 14th and 16th week
What is chorionic villus sampling?
Removing cells from the chorion with fetal tissue to examine for any disorders
When is CVS done?
Between 10th and 13th week
When can sample for karyotyping be obtained from?
Peripheral blood
Bone marrow biopsy
What is the most reliable examination to determine the sex chromosomes?
Blood sample from peripheral blood
What are the limitation of karyotyping ?
Can only detect major structural abnormalities, affect the whole chromosome
Labor intensive
Depends on experience and skill
What is Souther Blot Analysis used for?
Detection of a specific DNA sequence in DNA samples
What is the principle of method of Southern Blot?
Hybridization process; single strand probe which contains fluorescence.
If it binds, there will be a signal released which releases fluorescence and signals that the patient does have that DNA sequence
What is the process of Southern Blot?
Isolation of genomic DNA
Cleave with restriction enzymes
Transfer to nitrocellulose membrane
Hybridise with fluorescent or radiolabeled DNA probe
What are the advantages of Southern Blot?
Can detect point mutations that disrupt restriction sites
Larger structural changes (deletion, insertions and translations)
How are point mutations that disrupt restriction sites detected with Southern Blot?
If there is a change in the restriction site it will not be cut by the restriction enzyme
What is the major limitation of Southern Blot?
Requires large amounts of input DNA
What is PCR?
An in-vitro technique used for amplification of a region of DNA
What is a characteristic that needs to be present for PCR to work?
The sequence of DNA needs to be known or the sequence needs to lie between two regions of known sequences
What are the reaction components for PCR?
DNA template
Primers
Enzyme
dNTPs
Mg2+
Appropriate buffers
Why does the sequence or regions around it need to be known for PCR to work?
If the sequence or regions are not known, primers cannot be created so PCR cannot happen
How many primers are required for PCR and what are they ?
Two; one forward and one reverse
What is a difference between normal replication and PCR?
In normal replication, primers are RNA
Why do primers need to be about 2 to 35 nucleotides in length?
If larger they might produce internal structures so no hybridisation
Why does the enzyme need to be thermostable?
So that it can only denature at really high temperatures, 95oC
Why is Mg2+ used?
Acts as a cofactor and catalyser in PCR
What is the importance of buffer solution?
Provides suitable chemical environment for optimum activity and stability
What are the basic concepts of PCR?
Thermal cycling, heating up and cooling down
What is the importance of thermal cycling?
Causes DNA melting and enzymatic replication of the DNA
What are the three steps of PCR?
Denaturation
Hybridisation (annealing)
DNA synthesis
What are the differences between Reverse PCR and normal?
Uses RNA as an initial template instead of DNA
RNA directed DNA polymerase used
Yields ds cDNA
cDNA then used for standard PCR analysis
Where is PCR used?
Diagnostics
Genome mapping
Forensic
Detection of drug resistance genes
What are the advantages of PCR?
Automated, fast, reliable
High specificity and sensitivity
Broad uses
Easy to follow
What is a disadvantage of PCR?
Need to be careful and specific to not cause impurities
What does FISH detect?
Small deletions and replications
Examples of what FISH can detect:
Translocation
Deletion
Inversion
Isochromosome
Insertion
Ring chromosome
Derivative chromosome
FISH procedure:
Slides prepared from cultured (metaphase) or uncultured (interphase) cells
Fixed cells exposed to probe
Chromosomes and probe denatured
Hybridisation
Fluorescence staining
Where is sample for FISH taken from?
Peripheral blood
Fibroblasts
Epithelial cells
Bone marrow
Solid tumor
What are some applications of FISH?
Prenatal and cancer diagnosis
Molecular cytogenetic of birth defects
Specific chromosome abnormality
Characterisation of marker chromosomes
Monitoring bone marrow transplantation
Limitations of FISH:
Cannot identify the chromosomal abnormalities other than those detected by the specific probe
Preparation is critical
Unable to detect small mutations
Probes are not commercially available for all chromosome regions
Expensive
Limitations of microarray?
Complex data, hard to interpret
Expensive
