Factors determining primer-template annealing in PCR:

• Primer nucleotide sequence determines complementary hydrogen bonding and melting temperature ($T_m$).
• Annealing temperature directly controls hybridization stringency.
• Taq DNA polymerase specificity influences extension fidelity and processivity during elongation, but does not affect the thermodynamic hybridization of primers to template strands.
• Template GC content affects secondary structures and strand dissociation.
• Divalent $\text{Mg}^{2+}$ ions shield negative charges on DNA phosphate backbones, greatly affecting annealing kinetics and stability.

Verification of amplicon specificity in qPCR:

• Cycle threshold ($C_t$) reflects initial template quantity, not sequence specificity.
• Product sequencing is an independent downstream verification method, not an integral step of real-time PCR run analysis.
• Fluorescence intensity reflects accumulated double-stranded DNA during SYBR Green assays, including non-specific products.
• Melt curve (dissociation curve) analysis measures fluorescence decay as temperature rises slowly post-amplification; specific amplicons produce a distinct, sharp melting peak at their characteristic melting temperature ($T_m$), easily differentiating them from primer-dimers and non-specific products.