Real-time polymerase chain reaction

Real-time Polymerase Chain Reaction

Diagram of qPCR cycling process

Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR (qPCR), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e., in real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively (quantitative real-time PCR) and semi-quantitatively (semi-quantitative real-time PCR).

Principles of Real-time PCR

Real-time PCR is based on the detection and quantification of a fluorescent reporter. The signal increases in direct proportion to the amount of PCR product in a reaction. The most common real-time PCR chemistries are based on the use of fluorescent dyes that intercalate with double-stranded DNA, or on sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

Fluorescent Dyes

Melting curve analysis in qPCR

The most commonly used fluorescent dye is SYBR Green, which binds to double-stranded DNA. As the PCR product accumulates, the fluorescence increases. However, SYBR Green can bind to any double-stranded DNA, including primer-dimers, which can lead to non-specific signals.

Sequence-specific Probes

Sequence-specific probes, such as TaqMan probes, Molecular Beacons, and Scorpion probes, provide a higher specificity. These probes are labeled with a fluorescent reporter at one end and a quencher at the other end. During the PCR, the probe is cleaved by the 5' to 3' exonuclease activity of Taq polymerase, separating the reporter from the quencher and resulting in an increase in fluorescence.

Molecular Beacons used in qPCR

TaqMan Probes

TaqMan probes mechanism

TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. They are oligonucleotides that contain a fluorescent dye at the 5' end and a quencher at the 3' end. When the probe is intact, the proximity of the quencher reduces the fluorescence emitted by the dye. During PCR, the probe is cleaved by the polymerase, separating the dye from the quencher, resulting in an increase in fluorescence.

Molecular Beacons

Molecular Beacons are hairpin-shaped probes that fluoresce upon hybridization to their target sequence. They are designed to remain non-fluorescent when free in solution, but become fluorescent when bound to their target.

Applications of Real-time PCR

Real-time PCR is used in a variety of applications, including:

• Quantification of gene expression: By measuring the amount of mRNA, researchers can determine the level of gene expression in different tissues or under different conditions.
• Pathogen detection: Real-time PCR is used to detect and quantify pathogens in clinical samples, such as viruses, bacteria, and fungi.
• Genotyping: It is used to detect single nucleotide polymorphisms (SNPs) and other genetic variations.
• DNA quantification: It is used to quantify DNA in various samples, such as forensic samples or environmental samples.

Melting Curve Analysis

Melting curve analysis example

Melting curve analysis is a technique used to verify the specificity of the PCR products. After the amplification phase, the temperature is gradually increased, and the fluorescence is measured. As the double-stranded DNA melts, the fluorescence decreases. The melting temperature (Tm) is characteristic of the specific DNA sequence, allowing for the identification of specific products and the detection of non-specific products such as primer-dimers.

Related Pages

• Polymerase chain reaction
• DNA sequencing
• Gene expression
• Fluorescent dyes