Commonly Used Dyes
Although probes have the advantage of added specificity, dyes are more commonly used because of convenience and cost. Furthermore, PCR product melting analysis largely equalizes the specificity difference between dyes and probes.
Melting analysis of SYBR Green I, first introduced with the LightCycler in 1997, is now widely used on all instruments, although it is sometimes rebranded as dissociation analysis. The advantage of melting as an analytical technique is that, just like real-time PCR, it requires fluorescence detection and temperature control and can be performed in the same instruments.
More recently, melting analysis has become even more powerful as its resolution has increased. High-resolution DNA melting analysis (also known as HRM) is now accepted as the best variant scanning method and the simplest method for genotyping.
Genotyping is performed by bracketing the variation with a small PCR product that has a melting curve shape and/or position that depends on the genotype. Variant scanning is performed with dyes that detect heterozygotes (saturation dyes) as a change in melting curve shape.
When first introduced, melting analysis in conjunction with PCR was a simple check for specificity summarized by its melting temperature, or Tm. A pure PCR product was expected to have a single transition and a characteristic Tm.
This simple assumption turned out to be wrong for many PCR products; multiple domains are often detected with high-resolution melting and can be accurately predicted. This increased precision of DNA melting provides an analytical tool that is replacing gel electrophoresis for many applications.
In addition to PCR product melting, probe melting analysis is possible without fluorescently labeled probes. Using the same dyes that detect PCR products, the melting transitions of unlabeled probes or internal secondary structure can be monitored. Unlabeled probes require a 3´-end block to prevent extension, but otherwise avoid any covalent modifications.
If unlabeled probes are attached as 5´-tails to primers, they do not require blocking at the 3´-end. After PCR, these, “snapback primers” form intramolecular hairpins with the melting of the stem sensitive to the internal sequence. When unlabeled probe or snapback primers are included in PCR, both product scanning and specific genotyping can be performed.
Part of the appeal of real-time PCR and melting analysis is to integrate previously separate amplification and analysis steps without large-scale automation. Further integration of sample preparation into small diagnostic devices has now been FDA approved by at least two companies, the GeneXpert (Cepheid) and the FilmArray (Idaho Technology).
The FilmArray, for example, achieves sample disruption (bead beating), nucleic acid purification, reverse transcription, multiplex PCR for preamplification of 15–50 targets, secondary individual PCRs on a mini-array, and high-resolution melting analysis, all in less than one hour.
In summary, we have come a long way in making PCR faster, cheaper, and better. This article is necessarily biased by my own experiences, and my apologies if I have left out your favorite PCR derivative or direction. The fact that there are so many supports the power, flexibility, and value of the fundamental method. PCR will remain as one of the basic tools of the genetic engineer as an elegant and robust method for targeted amplification.
What of the future? The speed of PCR and melting is still limited by temperature measurement and control in commercial instruments. Temperature protocols are not transferable between manufacturers and most users typically think under the equilibrium temperature/time paradigm rather than the kinetic reality of constantly changing temperature necessary for rapid PCR.
Users and manufacturers have, in the past, been more concerned about numbers of samples (batch size) than speed and quality of the reactions. This historical choice of quantity over quality provides an opportunity going forward to improve temperature control, and thereby, speed and reproducibility between samples and instruments.
The potential of high-quality PCR and melting in less than 10 minutes is real. Other areas open to further progress are design and prediction tools focused on PCR. Although many primer and probe designs are available, few if any of the commonly accepted rules are based on empirical evidence.
Prediction tools for Tm are often inaccurate and complicated by the unfortunate trend of manufacturers to include proprietary ingredients that are not disclosed and users’ willingness to purchase them. Perhaps it is another tribute to PCR that it works so often given the current limitations of the state of the art.