Molecular Biology: PCR Process

PCR Process

• The PCR process consists of three major steps, which are repeated approximately 30 times.

Denaturation

• The first step is denaturation, where the template DNA is heated up to break apart the double-stranded DNA into two separate strands.

Annealing

• The second step is annealing, where the temperature cools down, allowing the primer set to bind to a specific area of the template DNA.

Extension

• The third step is extension, where a DNA polymerase pulls in bases (dNTPs) to create a single-strand copy of the template DNA.

5' Nuclease Assay Process

• The 5' nuclease assay process utilizes the 5'-3' exonuclease activity of Thermus aquaticus (Taq) polymerase.
• The process involves cleaving a dual-labelled probe annealed to a target sequence during amplification.
• The release of a fluorogenic tag from the 5' end of the probe is proportional to the target sequence concentration (copy number).
• The fluorogenic tag release can be measured in two ways:
  • Endpoint measurement (post-amplification)
  • Real-time measurement, where the increase in emission intensity is followed on a per-cycle basis.

TaqMan® Probe and 5' Nuclease Assay

• The TaqMan® probe anneals specifically to a complementary sequence between the forward and reverse primer sites during PCR.
• The 5' nuclease assay process takes place during PCR amplification, occurring in every cycle without interfering with the exponential accumulation of product.

TaqMan® Probe Structure and Function

• The TaqMan® probe contains a 5' reporter dye and a 3' quencher dye.
• When both dyes are bound, the reporter fluorescence is suppressed by the Fluorescent Resonance Energy Transfer (FRET) principle.
• Taq DNA polymerase enzyme cleaves only probes that are hybridized to the target, separating the reporter dye from the quencher and increasing fluorescence.

Signal Generation and Nonspecific Amplification

• The increased fluorescence signal occurs only if the target sequence is complementary to the probe and is amplified during PCR.
• Nonspecific amplification is not detected due to the requirements of probe hybridization and target sequence complementarity.
• Following probe cleavage, polymerization of the strand continues, but the 3' end of the probe is blocked, preventing its extension during PCR.

Learn about the three major steps of Polymerase Chain Reaction (PCR) process, including denaturation, annealing, and extension.