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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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High Resolution PCR Quantitation by AmpliSensor Assay.

¹Biotronics Corporation, Lowell, MA.

Methods in Molecular Medicine

|February 23, 2011

Summary

The AmpliSensor assay offers real-time polymerase chain reaction (PCR) quantitation by monitoring primer depletion during amplification. This method enables precise measurement of PCR products through a novel two-step amplification process.

Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Accurate quantification of Polymerase Chain Reaction (PCR) products is crucial for various molecular biology applications.
Existing PCR quantitation methods may have limitations in real-time kinetic analysis.
The need for sensitive and precise tools for monitoring amplification dynamics is ongoing.

Purpose of the Study:

To introduce and characterize the AmpliSensor assay as a novel real-time kinetic tool for PCR quantitation.
To demonstrate the mechanism of the AmpliSensor assay in quantifying PCR products.
To evaluate the performance of the AmpliSensor assay for accurate amplification monitoring.

Main Methods:

The AmpliSensor assay utilizes a two-step amplification strategy.

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Step 1 involves an initial amplification with an excess of one primer to overproduce a single strand of the target DNA.

Step 2 employs a subsequent hemi-nested amplification coupled with fluorescence signal detection for product quantitation.

Main Results:

The AmpliSensor assay quantifies PCR product by measuring the rate of the amplification reaction.
Quantitation is achieved through the progressive depletion of a rate-limiting primer during the reaction.
The two-step amplification scheme allows for sensitive and real-time monitoring of PCR product formation.

Conclusions:

The AmpliSensor assay provides a robust real-time kinetic method for PCR product quantitation.
This assay leverages primer depletion kinetics for accurate amplification measurement.
The AmpliSensor assay offers a valuable tool for molecular diagnostics and research requiring precise PCR quantification.