Laboratory Genetics and Molecular Diagnostics
Molecular and genetics questions test whether you can choose the right test, control the pre-analytical phase, and report a variant with an honest level of certainty.
Core rules
- Sample type: EDTA whole blood for germline DNA, plasma for cell-free DNA, formalin-fixed paraffin-embedded tissue for somatic testing; record tumour cellularity (often at least 20%) because it sets the detectable variant allele fraction.
- PCR detects nucleic acid, not living organisms; a positive result after treatment may reflect non-viable DNA.
- Real-time PCR quantification uses Ct/Cq values inversely related to target amount; each assay needs a validated cut-off and limit of detection.
- Karyotyping detects balanced rearrangements and aneuploidy at roughly 5–10 Mb resolution; FISH targets specific loci; chromosomal microarray detects copy-number gains and losses but not balanced translocations.
- Sanger sequencing suits single or few amplicons; next-generation panels require defined coverage (commonly ≥100× for germline, higher for somatic) plus review of strand bias and variant allele fraction.
- HGVS nomenclature uses c. for coding DNA and p. for protein, with the reference transcript stated.
- Variant classification follows the ACMG/AMP five-tier scheme: pathogenic, likely pathogenic, variant of uncertain significance, likely benign, benign. A classification is not a clinical diagnosis.
- Report only what is validated and consented; germline results have implications for relatives, somatic results guide therapy.
- Controls each run: positive, negative, extraction blank and no-template control.
Worked example
A heterozygous nonsense variant (c.1207C>T, p.Arg403Ter) is found in a gene where haploinsufficiency is an established mechanism. It is absent from population databases, the phenotype fits, and parental testing shows it is de novo. A reasonable classification is likely pathogenic, citing PVS1, PM2 and PS2, with the phenotype-specific evidence stated. The report should show the criteria applied rather than simply announcing that a mutation was found.
Traps
- Reporting a variant of uncertain significance as pathogenic and prompting surgery.
- Ignoring population allele frequency, which differs in impact between dominant and recessive disease.
- Applying a somatic assay's limit of detection to a germline question.
- Cross-sample contamination producing a false heterozygous call.