Molecular Techniques and Assay Design
Concepts to master
Know the architecture of PCR (denaturation, annealing, extension), real-time chemistries (TaqMan hydrolysis probes, molecular beacons, SYBR intercalation, scorpions), and the meaning of Cq/Ct. Understand multiplexing constraints (primer-probe competition, limited dNTP/enzyme, spectral overlap of fluorophores), and the design rules for primers and probes (length, GC content, Tm matching, avoidance of SNPs under the 3' end, amplicon size).
Beyond PCR
Be able to compare amplification methods (LCR, TMA, NASBA, SDA, HDA), hybridization formats (Southern/Northern, dot blot, FISH, microarrays), and sequencing generations: Sanger (dideoxy, capillary, ~800 bp reads), short-read NGS (bridge or emulsion amplification, sequencing-by-synthesis, reversible terminators), and long-read platforms (single-molecule, native DNA, better for structural variants and repeat expansions). Understand library prep, indexing/barcoding, target enrichment (hybrid capture vs amplicon), and the trade-offs in coverage, depth, and error modes (substitution vs homopolymer errors).
Traps
Questions confuse analytical specificity with clinical specificity, conflate limit of detection with limit of blank, and misattribute probe-based fluorescence to intercalating dyes. Watch for items where a multiplex assay fails because of primer-dimer, or where a low-level variant is missed because of allele dropout or a probe binding-site polymorphism.
Rule of thumb
When designing a diagnostic assay, match the chemistry to the question: quantitative viral load needs a calibrated real-time assay with an internal control; a structural rearrangement needs a method that spans the breakpoint (FISH, long-read, or breakpoint PCR), not a short-read SNP panel. Always include an internal amplification control and a no-template control.
Worked example
A quantitative HBV assay reports a Cq of 28 for a sample and 24 for a 10^6 IU/mL calibrator. With a slope implying one Cq per ~3.3-fold change (efficiency ~100%), a 4-Cq difference corresponds to ~10^1.2, or roughly a 1.2-log lower titer (~4×10^4 IU/mL), pending the standard curve.