Genetic Testing Technologies and Methodologies
Core concepts
Candidates must know the main assay families and what each is designed to detect: PCR/Sanger sequencing (single-site or short-fragment, gold standard for confirming a known variant), qPCR and MLPA (copy-number dosage at targeted loci), karyotyping (balanced and unbalanced structural change, ~5–10 Mb resolution), FISH (locus-specific, rapid, on interphase or metaphase cells), chromosomal microarray/CMA (genome-wide copy number and absence of heterozygosity at kb resolution; does not detect balanced rearrangements), next-generation sequencing panels, exomes and genomes, and expansions assays (repeat-primed PCR, Southern blot).
Understand the trade-offs: breadth of coverage versus depth, detection of mosaicism, turn-around time, and the variant classes each method cannot see. Recognize that orthogonal confirmation is expected for clinically actionable findings, and that the appropriate first-tier test depends on the clinical question (e.g., CMA first-tier for developmental delay; targeted variant testing first when a familial variant is known).
Traps
Questions often present a scenario where the wrong assay is chosen for the variant class - e.g., expecting microarray to detect a balanced translocation, or Sanger to detect a large deletion. Watch for confusion between analytic sensitivity (can the assay see it) and clinical sensitivity (does the test answer the question). Also beware of assuming a negative panel excludes all disease; panels only interrogate covered regions.
Rule of thumb
Match the test to the lesion: sequence change → sequencing; copy number → dosage method (CMA/MLPA/qPCR); position/structure → cytogenetic method (karyotype/FISH); repeat expansion → expansion-specific assay.
Worked example
A child with developmental delay and dysmorphism: first-tier CMA detects a 400 kb deletion. A balanced Robertsonian translocation in a parent would not be seen by CMA - a karyotype would be required.