To perform Real-time PCR, the extracted nucleic acid (NA) is combined with individual reagent components necessary to drive the reaction. These components consist of the NA extract along with primers, dNTPs (purine and pyrimidine bases), and a polymerase enzyme (most commonly-used Taq polymerase). The mixture is optimized with buffer and additional reagents to stabilize and drive the reaction.
Description of reagents:
- Primers – Short fragments of oligodeoxynucleotides, 18-25 bps in length, that flank the target sequence.
- Thermostable Polymerase – Polymerase is responsible for extension. Commonly used is the thermostable Taq polymerase.
- Enzyme Cofactors – Works with the polymerase, generally magnesium.
- Free dNTPs – The actual building blocks for DNA.
- Stabilizer – Includes buffer, glycerol, KOH, etc. stabilizes the reaction.
