Nucleic Acid Amplification, Hybridization, and Detection Methods
Amplification chemistry
PCR requires template, primers, dNTPs, Mg2+ as polymerase cofactor, buffer, and a thermostable polymerase. Cycling is denaturation near 95 °C, annealing about 5 °C below primer Tm, and extension near 72 °C. Hot-start enzymes reduce mispriming. Real-time chemistries include intercalating dyes (SYBR Green, requiring melt-curve confirmation), hydrolysis probes (TaqMan, using 5'→3' exonuclease activity), FRET hybridization probes, molecular beacons, and Scorpions. Multiplexing requires probes with distinct fluorophores and carefully balanced primers to avoid competition and dimer formation.
Quantitation
Ct (Cq) is inversely proportional to starting target: one cycle equals a two-fold difference, and 3.32 cycles equals ten-fold. Efficiency is derived from the standard-curve slope, E = 10^(−1/slope) − 1, with a slope of −3.32 representing approximately 100%. Digital PCR partitions reactions and gives absolute counts without a standard curve. Isothermal platforms (LAMP, TMA, NASBA, SDA, HDA) suit low-resource or high-throughput settings; branched DNA and hybrid capture amplify signal rather than target.
Hybridization and detection
Stringency increases with temperature and decreases with salt or formamide; wash conditions set specificity. Readouts include capillary electrophoresis, melt analysis and high-resolution melting, microarrays, MALDI-TOF mass spectrometry, Sanger sequencing, and next-generation sequencing.
Traps
Wrong answers usually reverse the Ct relationship, treat a one-cycle difference as ten-fold, or ignore post-amplification contamination. Amplicon carryover is controlled with dUTP/UNG systems, unidirectional workflow, and bleach.
Worked example
Standards at 10^6 and 10^3 copies give Ct 22 and 32; ten cycles across three logs yields slope −3.33 and efficiency near 100%.