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.