Real time PCR

Real-time PCR

Real-time PCR, also referred to as quantitative PCR or qPCR, is a sophisticated molecular biology technique designed for the accurate quantification of nucleic acids. Unlike conventional PCR, real-time PCR amplifies DNA, cDNA, and RNA targets while simultaneously monitoring the amplification process through fluorescence, enabling precise measurement of the starting material amounts. Fluorescence is measured during each cycle, which greatly increases the reaction's dynamic range since the amount of fluorescence is proportional to the amount of PCR product. PCR products can be detected using either fluorescent dyes that bind to double-stranded DNA or fluorescently labeled sequence-specific probes.

SYBR Green I is a fluorescent dye used extensively in real-time PCR assays to detect and quantify double-stranded DNA. It binds to all double-stranded DNA molecules, emitting a bright fluorescent signal upon binding (see figure SYBR Green principle). The excitation and emission maxima of SYBR Green I are at 494 nm and 521 nm, respectively, compatible with virtually any real-time PCR machine.

In SYBR Green PCR, the detection occurs during the extension step of real-time PCR, where signal intensity increases with each cycle due to the accumulation of PCR products. This method allows for analyzing many different targets without synthesizing target-specific labeled probes. However, it's important to note that nonspecific PCR products and primer dimers can also contribute to the fluorescent signal, thus necessitating high PCR specificity when using SYBR Green I. This characteristic underscores the need for precise primer design and reaction setup to ensure accurate results.

SYBR Green principle
Fluorescently labeled probes

Fluorescently labeled probes provide a highly sensitive method of detection, as only the desired PCR product is detected. However, PCR specificity is also important when using sequence-specific probes. Amplification artifacts such as nonspecific PCR products and primer–dimers may also be produced, which can result in reduced yields of the desired PCR product. Competition between the specific product and reaction artifacts for reaction components can compromise assay sensitivity and efficiency. The following probe chemistries are frequently used.

TaqMan probes

TaqMan probes are a type of sequence-specific oligonucleotide probe used in PCR equipped with a fluorophore at the 5' end and a quencher moiety at the 3' end. The fluorophore is attached at the 5' end of the probe, and the quencher moiety is located at the 3' end. During the combined annealing/extension phase of PCR, the probe is cleaved by the 5'–3' exonuclease activity of Taq DNA polymerase, separating the fluorophore and the quencher moiety. This results in detectable fluorescence proportional to the amount of accumulated PCR product

FRET probes

FRET probes are specialized tools used in PCR to enhance specificity and sensitivity via fluorescence resonance energy transfer (FRET). In this method, PCR employs two labeled oligonucleotide probes that bind to the PCR product adjacently in a head-to-tail fashion. When the two probes bind, their fluorophores come into close proximity, allowing energy transfer from a donor fluorophore to an acceptor fluorophore. Therefore, fluorescence is detected during the annealing phase of PCR and is proportional to the amount of PCR product. As the FRET system uses two primers and two probes, good design of the primers and probes is critical for successful results.

Fluorogenic probe dyes

Fluorogenic probe dyes in real-time PCR are critical components that enable the detection and quantification of specific DNA sequences during amplification. These dyes vary widely, each possessing unique excitation and emission maxima, which are essential for the specific identification of target DNA sequences (see table Dyes commonly used for quantitative, real-time PCR). The wide variety of dyes makes multiplex, real-time PCR possible (detection of 2 or more amplicons in the same reaction), provided the dyes are compatible with the excitation and detection capabilities of the real-time cycler used and the emission spectra of the chosen dyes are sufficiently distinct from one another. Therefore, when carrying out multiplex PCR, using dyes with the widest channel separation possible is the best practice to avoid any potential signal crosstalk.

Other probes

Many probe suppliers have developed their own proprietary dyes. For further information, please refer to the web pages of the respective suppliers.

Dyes commonly used for quantitative, real-time PCR
Dye Excitation maximum (nm)   Emission maximum (nm)*
Fluorescein 490 513
Oregon Green 492 517
FAM 494 518
SYBR Green I 494 521
TET 521 538
JOE 520 548
VIC 538 552
 Yakima Yellow 526 552
HEX 535 553
Cy3 552 570
Bodipy TMR 544 574
NED 546 575
TAMRA 560 582
Cy3.5 588 604
ROX 587 607
Texas Red 596 615
LightCycler Red 640 (LC640) 625 640
Bodipy 630/650 625 640
Alexa Fluor 647 650 666
Cy5 643 667
Alexa Fluor 660 663 690
Cy 5.5 683 707