Real time PCR

Real-Time PCR

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.

Principles of Real-Time PCR

Real-time PCR is used to quantitatively measure the amount of a specific DNA or RNA sequence in a sample. The technique relies on the detection of a fluorescent signal that increases in direct proportion to the amount of PCR product in a reaction. This is achieved through the use of fluorescent dyes or probes that bind to the DNA.

Fluorescent Dyes

Fluorescent dyes, such as SYBR Green, bind to double-stranded DNA. As the DNA is amplified, the dye binds to the newly synthesized DNA, and the fluorescence increases. This method is simple and cost-effective but can sometimes lack specificity.

Fluorescent Probes

Fluorescent probes, such as TaqMan probes, are more specific. These probes are oligonucleotides with a fluorescent reporter dye attached to one end and a quencher dye attached to the other. During PCR, the probe is cleaved by the DNA polymerase, separating the reporter and quencher, resulting in an increase in fluorescence.

Applications of Real-Time PCR

Real-time PCR is widely used in various fields, including:

• Medical Diagnostics: For detecting and quantifying pathogens, such as viruses and bacteria, in patient samples.
• Gene Expression Analysis: To measure the expression levels of genes in different tissues or under different conditions.
• Genotyping: For identifying genetic variations, such as single nucleotide polymorphisms (SNPs).
• Food Safety: To detect and quantify foodborne pathogens.

Advantages of Real-Time PCR

• Quantitative: Provides quantitative data on the amount of target DNA or RNA in a sample.
• Sensitive: Can detect low levels of nucleic acids.
• Specific: High specificity when using probes.
• Rapid: Faster than traditional PCR methods.

Limitations of Real-Time PCR

• Cost: More expensive than conventional PCR due to the need for specialized equipment and reagents.
• Complexity: Requires careful optimization and validation of assays.

Also see

• Polymerase Chain Reaction
• Gene Expression
• Fluorescent Dyes
• DNA Sequencing