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 (e.g., 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: Generally, magnesium is used as a cofactor working with the polymerase. The magnesium ion (Mg2+) works with the polymerase to enable incorporation of the dNTPs during polymerization.
- Free dNTPs: dNTPs are deoxynucleotide triphosphates which working with the Taq polymerase, are used to expand the growing DNA strand. The dNTPs are the actual building blocks for DNA.
- Stabilizer: Includes buffer, glycerol, KOH, etc. used to stabilize the reaction.
- Detection of PCR Products: Real-time PCR typically uses fluorescent dyes or fluorescence-containing DNA probes to measure the amount of product as the amplification progresses.
Note: Descriptions of reagents and components of the real-time PCR method are discussed in more detail in other sections.
