Construction of polycompetitors for competitive PCR

Area of Science:

• Molecular Biology
• Biotechnology

Background:

• Competitive polymerase chain reaction (PCR) requires specific mimics or competitors for accurate quantitation.
• The design and construction of these reference molecules are critical for protocol success.

Purpose of the Study:

• To provide a detailed protocol for developing individual mimics and polycompetitors for competitive PCR.
• To enable the creation of a single reagent (polycompetitor) with multiple specificities for simultaneous analysis.

Main Methods:

• Utilizes detailed restriction-endonuclease mapping for primer design and construct incorporation.
• Employs a specific cloning vector (pGEM 11Z) and outlines steps for constructing a human T-cell cytokine polycompetitor (pDC10).

Main Results:

• Demonstrates a successful method for producing cytokine polycompetitors for both human and mouse.
• The protocol is adaptable for creating polycompetitors for virtually any molecule of interest with available sequence data.

Conclusions:

• The described protocol offers a robust and generalizable method for constructing competitive PCR reagents.
• Careful planning and utilization of restriction maps are key for successful polycompetitor synthesis.