1-43 Genetic Diagnostic Technologies Flashcards
What is FISH, and what are some types of FISH analysis?
Fluorescence in situ hybridization, a targeted assay. Molecular probes (i.e., a fragment of DNA) labeled with a fluorochrome are hybridized to chromosomes, producing results that are quickly examinable with a fluorescent microscope. The most common goal is to determine if a gene, a specific mutation, or a particular chromosomal rearrangement is present or absent, so the molecular probe(s) used must be well characterized and specific to the locus being examined. Some types of FISH analysis include:
- Repeat sequences
- Single copy DNA (incl. subtelomere FISH): the most helpful method today; used for single copy, site-specific gain/loss of DNA
- Chromosome painting (incl. multi-color)
What is the technical process for FISH?
- Create a probe: choose a target (e.g., a DNA gene or location) and isolate a fragment of DNA (should be unique to this region). Label one strand of the fragment with a fluorescent dye.
- Confirm that the probe works: hybridize probe to a metaphase cell preparation; it should bind to the same location from which it was derived.
- Testing: FISH can be performed on metaphase OR interphase cells. Prepare slides just like for a karyotype analysis. Denature the DNA on the slides, and the fluorescently labeled single-stranded molecular probe is allowed to hybridize to the single-stranded chromosomal DNA.
- Post-testing: Following renaturation of the DNA, counterstain the rest of the DNA with another fluorochrome to allow visualization of the entire chromosome complement using a fluorescent microscope.
What are some benefits to FISH analysis?
- Easy to score using the colors
- Easy to score larger numbers of cells
- quickly
- Does not require metaphase cells (can use interphase, too)
- Assays the entire population
- Provides a more accurate estimate of the frequencies for different cell populations
What are some problems with FISH analysis?
- Probes do not cover the entire abnormality – just the “critical” region (e.g., for a 3 MB deletion, the probe may be only 10 KB)
- A very small duplication or deletion may be too small for the probe to hybridize
- A variant duplication or deletion may not be detected if the probe is outside of the boundaries of the abnormality
What are microarrays, and what are some types of microarray analysis?
A type of gene chip technology whose studies include hundreds to thousands of genes and are all performed on an area equal to or smaller than a standard microscope slide. Basic principle: compare a test DNA (one color) to a reference DNA (second color) that has a known genetic complement. After hybridization of the DNAs, the resulting fluorescent signals are identified and recorded, resulting in an array whose various combinations of the two colors can be analyzed to produce data about deletions (excess reference DNA) and duplications (excess test DNA). Some types of micro-arrays are:
- DNA (gene) arrays: looking specifically at DNA sequences of interest)
- Expression arrays (RNA): looking at gene products to understand which genes are being expressed in a particular cell type at a particular time
- Chromosome arrays: looking for abnormalities that are in some way related to the chromosomes
What can microarray analysis potentially identify?
Depending on the DNAs placed on the chip, the analysis can identify:
- Genetic polymorphisms or specific mutations
- Mosaicism
- Copy number variation (CNV; gain or loss of DNA that can be directly associated with genes or specific regions of the genome) > 10kb
- Absence of heterozygosity (AOH)
- Regions of homozygosity (ROH)
This type of assay will usually NOT detect balanced rearrangements because if the rearrangement is completely balanced, the total amount of DNA is conserved and no change in the relative amounts will be detected.
How do microarrays compare to karyotype analysis?
Microarrays are superior in detecting abnormalities for individuals with developmental delay, intellectual disability, autism spectrum disorders, and multiple congenital anomalies. Microarray analysis should be the first tier study in these types of cases, while karyotype analysis should be primary for patients with features of known chromosomal disorders or cases involving chromosome rearrangements.
What is IBD, and what are its associated risks?
Identity by descent. This category can include cases of consanguinity, incest, inbred populations (geographically isolated), and ethnic populations. Associated risks include:
- Increased risk of homozygosity for recessive, deleterious traits
- Increased risk of expression of autosomal recessive traits
- A high frequency of homozygosity has been associated with developmental delay and autism spectrum disorders
What genetic technology is particularly useful for patients with IBD?
Microarray analysis, because it can identify AOH and ROH.
What are some pros and cons to clinical use of next generation sequencing?
Pros:
- Targeted OR genome-wide screen
- Detection of very small abnormalities (_>_1 bp)
Cons:
- Cost
- Time for reporting
- Amount of data with no clinical correlation
- Difficulty in detection of mosaicism, translocations, UPD/IBD
Which genetic technologies are genome-wide? How do they compare to one another?
Karyotype analysis and microarrays.
Karyotype analysis works on relatively large numerical and structural abnormalities, while microarrays can screen for small to large mutations. Microarrays will not, however, detect balanced rearrangements.
Which genetic technologies are targeted? How do they compare to one another?
Molecular diagnostics, FISH, and DNA sequencing.
Both MD and FISH are well defined and specific, but MD targets very small mutations while FISH targets medium ones. DNA sequencing provides high resolution testing on the single base level.
How do microarray analysis and sequencing compare?
Although sequencing is more thorough than microarray, it can have a difficult time identifying copy number variants, mosaicism, uniparental disomy, and identity by descent, which are the backbone of the SNP microarray assay.
