= Solution
\b[PCR converts a selected <DNA> sequence into a detectable and manipulable quantity; different downstream measurements give it different uses.] A pair of <PCR primers> defines the interval amplified by <DNA polymerase>. Repeated denaturation, annealing and extension produce approximately $N_n=N_0(1+E)^n$ copies while <PCR efficiency> $E$ remains constant. At ideal efficiency, twenty cycles give about a millionfold amplification; depletion and accumulating products eventually cause a <PCR plateau>. Specificity derives from both primer-binding sites and their orientation, and should be checked by product size, melting behaviour or sequencing.
One application is <genotyping>. A disease-associated substitution can be detected by amplifying the surrounding region and reading the sequence, probing with an allele-specific oligonucleotide, or digesting a restriction site changed by the variant. An early demonstration amplified the human beta-globin region for restriction-based diagnosis of <sickle cell disease>. Amplification increased sensitivity; the restriction pattern supplied the allele information.<Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia by R. K. Saiki and others>[Saiki and colleagues, 1985, abstract: amplification followed by restriction-site analysis]{ref} A negative disease-specific signal is interpretable only if a control locus amplifies, because poor extraction or an inhibitor can mimic absence of an allele. In <forensic DNA profiling>, several <short tandem repeat> loci are amplified and fragment lengths compared. A multilocus match supports identity statistically; it does not eliminate contamination, mixtures, allelic dropout or the need for appropriate population-frequency estimates.
For infectious agents, primers can detect a pathogen-specific <DNA> sequence. An <RNA> virus first requires <reverse transcription>, followed by <polymerase chain reaction>. In <RT-qPCR>, fluorescence is monitored during amplification, and a threshold cycle is related to starting <RNA> using calibrated standards and efficiency measurements. This also measures changes in cellular <gene expression>: compare a target transcript between conditions after suitable normalization. Include extraction controls, a no-template reaction and, for <RNA> measurements, a no-reverse-transcriptase control to detect genomic <DNA> contamination. A positive pathogen signal establishes the presence of target nucleic acid, \b[not necessarily a viable organism or infectious virus]. Likewise, <RNA> abundance is not a direct measurement of <protein> abundance or transcription rate, since <RNA> degradation also matters.
PCR is also a preparative method. Amplified coding sequences can be assembled into expression vectors; primer tails can add restriction sites, assembly overlaps, sequencing adaptors or an affinity tag. High-fidelity polymerase reduces unwanted substitutions, but the final clone should still be sequenced. <Site-directed mutagenesis> introduces a specified substitution using mutagenic primers, whereas <error-prone PCR> creates a library for <directed evolution>. Sequencing after selection distinguishes the intended change from other mutations. In <chromatin immunoprecipitation>, quantitative amplification of recovered loci can test enrichment of a DNA-associated <protein>, with input <DNA> and immunoprecipitation controls needed to interpret it.
These examples separate presence, identity, quantity and construction. Endpoint band intensity is a poor substitute for quantitative amplification measured before the <PCR plateau>; multiplex assays can also suffer unequal amplification. The appropriate answer therefore identifies \b[what is amplified, what is measured, and which alternative explanation each control excludes].
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