Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Zachary A. Crannell; Brittany Rohrman; Rebecca Richards-Kortum

doi:10.3791/52620

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positiv...

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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

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08:37 min

March 30, 2015

DOI: 10.3791/52620-v

Zachary A. Crannell*¹, Brittany Rohrman*¹, Rebecca Richards-Kortum¹

¹Department of Bioengineering,Rice University

Overview

This article presents a protocol for a real-time recombinase polymerase amplification assay designed to quantify DNA concentrations in samples. The method utilizes either a thermal cycler or a microscope with a stage heater, and includes the development of an internal positive control.

Key Study Components

Area of Science

Biotechnology
Molecular Biology
Genomics

Background

Quantitative analysis of DNA is crucial in various biological research fields.
Traditional methods like real-time quantitative PCR require expensive thermal cyclers.
Recombinase polymerase amplification (RPA) offers an isothermal alternative.
Internal positive controls enhance the reliability of quantitative assays.

Purpose of Study

To develop a protocol for quantifying DNA concentrations using RPA.
To create an internal positive control for improved assay validation.
To provide scripts for analyzing real-time fluorescence data.

Methods Used

Preparation of reaction mixtures with target DNA, primers, and fluorescent probes.
Use of a real-time PCR machine to monitor fluorescence during amplification.
Analysis of fluorescent data to generate a standard curve.
Validation of the assay through experiments quantifying HIV target DNA.

Main Results

The assay accurately quantifies DNA concentrations within one order of magnitude.
Validation experiments confirmed the effectiveness of the internal positive control.
RPA demonstrated advantages over traditional PCR methods.
The protocol is accessible for researchers without expensive equipment.

Conclusions

The developed RPA assay is a reliable method for DNA quantification.
Internal positive controls are essential for assay validation.
This method can facilitate broader access to DNA quantification techniques.

Frequently Asked Questions

What is recombinase polymerase amplification?

Recombinase polymerase amplification (RPA) is a technique for amplifying DNA at a constant temperature, making it simpler and more accessible than traditional PCR methods.

How does the internal positive control work?

The internal positive control is a known DNA sequence added to the reaction to ensure that the assay is functioning correctly and to validate the results.

What are the advantages of using RPA over traditional PCR?

RPA is isothermal, meaning it does not require a thermal cycler, making it more cost-effective and easier to implement in various settings.

Can this method be used for other DNA targets?

Yes, the protocol can be adapted for various DNA targets beyond HIV, allowing for versatile applications in molecular biology.

What equipment is needed for this assay?

The assay can be performed using a real-time PCR machine or a microscope with a stage heater, making it accessible for many laboratories.

How is the fluorescent data analyzed?

Fluorescent data is analyzed using provided scripts that generate a standard curve for quantification and validation of the assay.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

The overall goal of the following experiment is to use quantitative recombinase polymerase simplification to quantify the DNA concentration of unknown samples. This is achieved by first adding the target DNA internal positive control, DNA primers and fluorescently labeled probes to the reaction. As a second step, the reactions are placed in the real-time PCR machine, which heats the reactions to activate the enzymes and monitors the fluorescence of the probes to detect the generation of target and control amplicons.

Next, the fluorescent data is analyzed using a script to generate the standard curve and to validate the assay. The results show that DNA samples can be quantified accurately within one order of magnitude of the correct concentration based on the validation experiments used to quantify an HIV one target DNA. The main advantage of this technique over existing methods like real-time quantitative PCR, is that RPA is isothermal, so an expensive thermal cycler is not needed.

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