Separation of the two strands of DNA is necessary for semiconservative replication and cell division (Meselson and Stahl, 1958). Through a complex enzymatic process, the DNA double helix unwinds and separates so that each strand can serve as a template for a new duplex. In vivo, enzymatic strand separation occurs under physiologic conditions of temperature, pH, and ionic strength. In vitro, double-stranded DNA can be converted to single strands chemically (alkali, low ionic strength, organic solvents, chaotropes) or physically (heat) and is often referred to as “DNA melting”. Heat is most convenient because it can be controlled precisely, providing a continuous gradient of increasing temperature. DNA melting or “denaturation” is a required step for the polymerase chain reaction (PCR). Temperature is used to sequence the steps of PCR between strand denaturation, primer annealing, and polymerase extension.

DNA melting can be used as an analytical technique. As the temperature changes, duplexed strands separate, a process conventionally monitored by absorbance at 260 nm (the hyperchromatic effect). Two separated single strands absorb more light than the compact structure of double stranded DNA. As DNA melts, its absorbance increases by 30–40% (Wartell ASB, 1985). The temperature at which half of the duplexes are melted is called the melting temperature, or Tm. The Tm depends on the GC content, length, and sequence of the duplex. Many short (<200 bp) duplexes melt in a single transition and the Tm can be approximated by the curve peak on derivative plots of absorbance vs temperature (Fig. 1). While electrophoresis gels characterize DNA by size, melting analysis segregates DNA according to Tm.

Only small, well-defined DNA duplexes melt in a single transition. It is more common for DNA to melt in multiple transitions, or domains. Domains with a greater proportion of GC base pairs melt at higher temperatures than domains with more AT base pairs. Typical domains are 50–500 bp in size. Multiple peaks are often observed on derivative plots of longer duplexes (>200 bp). The pattern of DNA domain melting can be used as a fingerprint, more powerful than the simple Tm of a short duplex, but not as discriminatory as sequencing. Many fundamental studies on DNA have been performed by absorbance (Britten DEK, 1968). Such studies typically require μg amounts of purified DNA, requirements not easily met with clinical samples.

Three factors have enabled DNA melting analysis to transition from a specialized research tool into routine research and clinical testing: 1) the advent of PCR to conveniently produce ng amounts of specific DNA duplexes, 2) the use of fluorescence rather than absorbance to increase sensitivity, and 3) instrumentation to acquire melting curves in minutes rather than hours. PCR amplifies a defined segment of DNA by over 107-fold (Saiki et al., 1988) from complex mixtures, such as human genomic DNA or microbial DNA admixed within a host environment. After amplification, most of the duplex DNA present is the targeted segment and fluorescence melting analysis can be performed in the presence of other absorbing species (dNTPs, primers). Fluorescence is also much more sensitive than absorbance; the amount of DNA produced by PCR is easily detected by fluorescence. SYBR Green I is most commonly used to stain the double-stranded DNA produced by PCR, although many different fluorescent dyes can be used (Gudnason et al., 2007). Finally, instead of slow melting rates using thick, glass cuvettes and 0.1–1.0 mL of solution that typically take hours, real-time PCR instruments (Wittwer et al., 1997a) use smaller volumes with rapid heat transfer for DNA melting in minutes. The combination of simple production (PCR), enhanced sensitivity (fluorescence) and faster analysis (instrumentation), make modern melting analysis a convenient and powerful research and diagnostic tool.

Here, we review advances in fluorescence melting curve analysis made possible by real-time PCR amplification. PCR product or “amplicon” melting is considered first, including high resolution melting, the introduction of saturating DNA dyes, high speed melting, highly parallel digital melting, and melting curve prediction. Probe melting is then considered, including adjacent hybridization probes, single hybridization probes, unlabeled probes and snapback primers. Research applications include amplification quality control, genotyping, variant scanning, methylation, copy number, and sequence identity. Finally, clinical microbiology applications are considered, including syndromic testing, point-of-care analysis, and population profiling.