GeneticsTier 1Medical Sciences concept

Molecular diagnostic techniques - PCR principles and applications

Core concept

PCR (polymerase chain reaction) amplifies a specific DNA segment via repeated cycles of denaturation (95C) -> annealing (primer binding, ~50-65C) -> extension (72C, Taq polymerase) -- exponential amplification
Primer specificity determines qPCR specificity: primers are designed to flank the target sequence -- poorly designed/non-specific primers cause off-target amplification and false results, independent of the underlying enzyme chemistry
Fragment sizing (e.g. for Huntington's disease) uses PCR to amplify the CAG repeat region then measures product size by capillary electrophoresis/fragment analysis -- repeat number determines fragment length, and thus disease status/risk

Correct PCR step sequence: denaturation -> annealing -> extension, repeated for ~25-35 cycles -- getting this order or the temperature logic wrong is a common exam distractor
Huntington's disease test of choice is PCR-based fragment/repeat-length sizing, not standard sequencing -- because the diagnostic question is how many CAG repeats, a length measurement, which capillary electrophoresis resolves precisely and cheaply
Whole exome sequencing (WES) covers only the protein-coding regions (~1-2% of the genome) -- misses regulatory/intronic/non-coding variants that whole genome sequencing (WGS) would capture; WES is preferred when disease-causing variants are expected in coding regions and cost/turnaround matter

Trap: ordering WES expecting it to detect a deep intronic or regulatory variant -- it will not, because WES simply does not sequence that DNA; WGS or targeted testing is required
Choosing fragment sizing over full sequencing for repeat-expansion disorders (Huntington's, fragile X, myotonic dystrophy) is deliberate -- sequencing struggles with very long/repetitive tracts, while fragment length assays measure them directly and reliably
Non-specific qPCR primers manifest as unexpected additional melt-curve peaks or unreliable quantification -- a lab quality-control issue tied directly to primer design, not instrument fault

Fragment-sizing methodology - see trinucleotide repeat disorders and genetic anticipation; same repeat-expansion biology, this note covers the testing method
WES/WGS coverage distinction is relevant whenever choosing a genetic test strategy for an undiagnosed condition -- feeds into variant pathogenicity assessment once a candidate variant is found
PCR is also the basis of many infectious disease diagnostics (e.g. viral load, TB NAAT) -- same amplification principle, different target

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