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PCR

PCR

PCR - Polymerase Chain Reaction

PCR - Polymerase Chain Reaction

Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

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Construction of polycompetitors for competitive PCR.

D B Corry¹, R M Locksley

¹Department of Medicine, University of California, San Francisco, CA.

Methods in Molecular Medicine

|February 23, 2011

Summary

This protocol details the creation of competitive polymerase chain reaction (PCR) mimics and polycompetitors. These reagents are essential for accurate molecular quantitation in various research applications.

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:

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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.