Real Time PCR

Real-time PCR is based on the generation of a fluorescent signal by the PCR process, which is detected during PCR cycling (i.e., in real time) and reflects the amount of PCR product synthesized.28-30 Different real-time PCR methods use alternative ways to generate a fluorescent signal during PCR. These include an intercalating dye such as SYBR Green that binds the minor groove of DNA, or an oligonu-cleotide used as a primer or probe and labeled with a fluorogenic dye. Instruments that combine in vitro nucleic acid amplification and real-time detection of the PCR product are dramatically increasing test menus for oncology, infectious diseases, and genetics in clinical molecular laboratories because of the wide range of readily available amplification primers and detection schemes, rapid turnaround time, the potential to eliminate the time and costs associated with electrophoresis, and the reduction in PCR contamination risk.

Real-time PCR is different from conventional PCR in several ways.

• Amplicon generation, temperature profiles, and melt curves are monitored in real time, reducing the time required for post-PCR analysis. In most cases, there is no need for postamplification processing of the PCR products. This eliminates the need for gel electrophoresis and, because the reaction tubes remain closed after PCR starts, there is decreased risk of amplicon carryover contamination within the laboratory.

• Results are more reproducible between runs since quan-titation of target is based on amplification cycle threshold in the log-linear phase of amplification rather than traditional endpoint analysis in the PCR plateau phase.

• Real-time PCR methods have a wide dynamic range, up to 10 logs.

• Real-time PCR systems with intercalating dye or fluoro-genic probes can be used to perform melting-curve analysis. This adds a check for specificity or potentially the detection of unknown sequence variants (see below).

The simplest real-time PCR method uses intercalating dyes that insert into the stacked bases of DNA PCR products, allowing detection of amplification in real time. These dyes, for example, SYBR Green and EtBr, are nonsequence-specific dyes that increase in fluorescence when bound to double-stranded DNA. Intercalating dyes are often used for melting-curve analysis, qualitative and semiquantitative PCR, product discrimination and purity, and determination of primer and probe melting Tm. Intercalating dyes can be used for quantitative PCR. Results, however, are more specific and accurate with a sequence-specific probe since fluorescence is directly proportional to the amount of specific amplicon produced and reduces the background contributed by primer-dimers or nonspecific PCR prod ucts. Intercalating dye fluorescence represents all double-stranded DNA, including primer-dimers and other nonspecific product that can be visualized with an endpoint melting-curve analysis.

Most fluorogenic oligonucleotide techniques take advantage of the principle of fluorescent resonance energy transfer (FRET), in which the energy from one dye molecule (the donor) is transferred without the emission of a photon to an acceptor dye molecule when the two are in close proximity. If the acceptor is a fluorophore, a photon is emitted at a characteristic wavelength. However, if the acceptor does not emit a photon, the energy is dissipated and fluorescence from the donor is quenched. The reporter dye can be either the donor (if no FRET takes place) or the acceptor (if FRET does take place) and is defined as the one whose fluorescence correlates with the quantity of desired PCR amplicon. Several fluorogenic techniques are described below.


TaqMan uses a short probe complementary to a non-primer internal sequence of the PCR product. The probe is labeled at the 5' end with a reporter donor dye and at the 3' end with an acceptor dye that quenches the reporter when the probe is intact. During the extension phase of PCR, probe bound to an amplicon is cleaved by the 5' endonuclease activity of Taq polymerase, freeing the reporter dye from the quencher and resulting in fluorescence. The fluorescent signal increases proportionally to the number of amplicons generated during the log-linear phase of amplification. To ensure that hydrolysis of the probe occurs, a two-step PCR can be used with annealing and extension taking place at the same temperature (~60°C). Ideally, the TaqMan probe binding site is located near one primer and the size of the amplicon is no longer than 200 to 300 bases. One negative aspect of this format is that once the probe is hydrolyzed, it is unavailable for subsequent reactions or melting-curve analysis, thus requiring an excess amount of probe in the reaction mix with the potential to decrease the PCR efficiency.

Molecular Beacon

A molecular beacon is a longer probe with a 5' reporter dye and 3' quencher dye. The probe forms a hairpin loop structure when not bound to target DNA, thereby juxtaposing the dyes with quenching of fluorescence. The loop sequence is complementary to the non-primer amplicon sequence. When the loop of the molecular beacon probe hybridizes to the amplicon during the annealing step of real-time PCR, the reporter dye is separated from the quencher, resulting in fluorescence. For the molecular beacon probe to anneal to the amplicon, the amplicon-probe hybrid must be more stable than the internal base-

pairing hairpin so that a fluorescent signal is generated. Generally, DABCYL is the nonfluorescent universal quencher and the other dye is a reporter fluorophore such as FAM, Cy3, TET, TAMRA, Texas Red, ROX, or Cy5.


Hybridization is a two-probe system in which one probe contains a donor dye and the other contains the reporter acceptor dye. The probes are designed to anneal to one strand of the PCR product adjacent to one another and internal to the primers. This juxtaposes the dyes, allowing FRET to occur. This probe format works well with the traditional three-step PCR with annealing at ~55°C (primer specific) and extension at 72°C, the optimal temperature for Taq polymerase activity. When DNA poly-merase encounters the probes, they are displaced from the target strand rather than hydrolyzed and thus available for the next round of amplification as well as endpoint melting-curve analysis.

Uniprimer (Amplifluor, Sunrise)

Like molecular beacon probes, the uniprimer system uses a hairpin structure in the oligonucleotide to quench fluorescence. The 3' region of the fluorogenic oligonu-cleotide is identical to a nonbinding region at the 5' end of the reverse PCR primer. This allows the fluorogenic oligonucleotide to become a primer for the newly formed amplicon by the third round of PCR. The probe is then opened in the fourth and subsequent rounds of PCR by the polymerase action of Taq, allowing fluorescence to occur. The advantage of this system is that the same fluorogenic oligonucleotide can be used in any PCR reaction (universal fluorogenic primer).


Scorpion also uses a hairpin structure in the oligonu-cleotide to quench fluorescence. The fluorogenic oligonu-cleotide is part of the reverse primer, and the nucleotides in the hairpin are complementary to the PCR amplicon sequence between the primers. The Scorpion primer unfolds and anneals to the PCR amplicon, allowing fluorescence to take place beginning in the first round of PCR.

Lux is a variation of real-time PCR that uses a single fluorophore in a primer with a hairpin loop structure. The fluorophore is quenched by the complementary structure of nucleotides in the stem of the hairpin loop. When the primer is incorporated into double-stranded DNA, thus opening the hairpin loop, fluorescence is maximal. The advantage of this system is lower production costs with the use of only one fluorophore.

Real-Time PCR Method

The following concepts are important for understanding the use of real-time PCR in a clinical diagnostic laboratory. When optimizing real-time PCR, the growth curve of the fluorescent signal versus the number of PCR cycles should be monitored to determine when optimal conditions have been achieved (Figure 2-2). The growth curve should be sigmoidal (S shaped) with three phases: baseline (background signal or lag phase), log-linear (exponential amplification phase), and plateau. For each phase, several characteristics should be assessed. The baseline phase of the curve represents initial cycles of amplification in which accumulation of the specific signal has not yet exceeded the background signal. The fluorescent signal in this phase is from unbound probe or autofluorescing components in the reaction. The log-linear phase of the curve represents exponential amplification of the target (see Figure 2-1) and provides useful information about the reaction. The curve can be described by the following equation: Tn = T0(E)n, where Tn is the amount of target sequence at cycle n, T0 is the initial amount of target sequence at cycle 0, and E is the amplification efficiency of the target sequence. The crossing point represents the number of PCR cycles at which the growth curve enters the log-linear phase. There is an inverse linear relationship between the crossing-point cycle number and the number of template copies present in a reaction.

The slope of the log-linear phase is a reflection of amplification efficiency, and the efficiency of the reaction can be determined by identifying the crossing points of known standards and plotting a line of linear regression (see Figure 2-3). The efficiency can then be determined using the following equation: E = io-1/slope, where E is efficiency and slope is the slope of the standard curve. Using this equation, the slope should be between -3 and -4, with -3.3 indicative of efficiency close to or at 2. The inflection point is the point at which the log-linear amplification curve goes from positive to negative and begins to enter the plateau phase. If there is no inflection point, the curve may represent not amplification of DNA, but rather signal drift. Drift is characterized by gradual increase or decrease in fluorescence without amplification of product.

Plateau is defined as the phase of growth when critical components become rate limiting and amplicon accumulation is minimized or stops. The plateau is also the point at which incremental increase in fluorescent signal stops. As the rate of accumulation slows and enters the plateau phase, the curve levels. Since endpoint measurements are often made in conventional PCR when reaction components are limited, minor sample variations can have a relatively major effect on endpoint product. Real-time PCR focuses on the rate of amplicon accumulation (log-linear phase and cycle threshold or crossing point), not on endpoint signal. The plateau phase can be shortened by decreasing the number of cycles for a product of greater purity. Several factors contribute to the plateau phase: PCR product reannealing versus primer annealing, enzyme or dNTPs becoming limiting, and amplicon buildup with resultant reaction inhibition.

Real-time PCR utilizes the log-linear phase of the amplification curve for data analysis. This method provides a more accurate measurement than endpoint analysis. The cycle at which the curve crosses a specified threshold is called the cycle threshold (Ct), or crossing point (Cp). The Ct value can be used for qualitative or quantitative analysis. A qualitative analysis uses the defined Ct as a pass/fail measurement. A quantitative assay uses the Ct of defined standards of known template concentration to generate a standard curve. Then the Ct values for unknown samples are used to extrapolate the concentration(s) in the unknown samples from the standard curve. Some real-time instrumentation software allows determination of the Ct by a mathematical analysis of the amplification curve, rather than crossing at a set fluorescent signal threshold. Plotting the second derivative of the growth curve generates a peak that corresponds to a point near the baseline of

















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Figure 2-2. Real-time PCR curves for a 10-fold dilution series of a known standard. Data provided by Roche Molecular Diagnostics. (Used by permission.Data © 2003 Roche Diagnostics Corporation, all rights reserved.)

Figure 2-2. Real-time PCR curves for a 10-fold dilution series of a known standard. Data provided by Roche Molecular Diagnostics. (Used by permission.Data © 2003 Roche Diagnostics Corporation, all rights reserved.)

Figure 2-3. Standard curve generated by real-time PCR of a 10-fold dilution series of a known standard.

Log concentration

the growth curve (see Figure 2-4). The cycle at which this peak occurs is designated as the Ct or Cp. This analysis method can provide better run-to-run reproducibility than manually setting the Ct using the primary signal.

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