Real Time PCR - Some Basic Principles

Several different chemistries are commonly used for real-time PCR detection. All of the systems have one principle in common; they all measure fluorescence during the PCR process. For all chemistries, the amount of emitted fluorescence is proportional to the amount of product (copies of the specific gene) being made in the PCR reaction.

The simplest principle of detecting fluorescence is based upon intercalation dyes that bind double-stranded DNA made during PCR. The other principles are based upon chemically modified oligonucleotides, or fluorescent labeled probes that are added during the setup of the reaction. Fluorescence is released and detected either after degradation of the probe (hydrolysis probes) or hybridization of two oligonucleotides to the target (hybridization probes). Adding the probe, along with the oligonucleotide primers increases the specificity of the quantified PCR product over the intercalation systems. Other detection technologies have also been developed. These include Scorpions (Whitcome et al., 1999; Thewell et al., 2000) and Molecular Beacons (Tyagi and Kramer 1996; Tyagi et al., 2000) but will not be discussed in this lesson. First we will discuss the intercalation system.

SYBR Green - Intercalation dye (Holden et al., 2003, Terry et al., 2002)
 

Figure 8. Diagram of SYBR Green detection system (Image adapted from: European Commission Joint Research Centre.)

SYBR green is an intercalating dye and functions similar to ethidium bromide (Figure 8). SYBR green binds more efficiently to double stranded DNA, than it does to single stranded DNA. Remember that after the heating cycles of PCR, the DNA molecules have been denatured and are single stranded. However, after the extension stage of PCR when the complementary copies of the templates have formed, a maximum amount of dye will bind into the minor groove of the now double stranded DNA. Thus, a fluorescence signal is detected and increases as more copies of the target gene are made at the completion of each cycle in the PCR process. Unfortunately, SYBR green will also bind to other double stranded, non-specific PCR products such as primer-dimers . To provide better sequence-specificity and detection, both FRET and Taqman technologies have been developed. These methods are discussed next.