Method for detection of nucleic acid targets by Amplification and Fluorescence Polarization

In the Public Domain since

15 Nov 2015

Description

In 1995, Linn Carl Preston et al., patented Fluorescent Polarization (FP), a method for detecting amplification of nucleic acid target sequences. This patent provides methods using FP and a signal primer comprising a fluorescent label for detection of nucleic acid amplification, particularly at elevated temperatures (e.g., at about 45°-75° C), where the speed and specificity of the target amplification are higher. Amplification is detected as an increase in FP associated with a target amplification-dependent generation of double-stranded, fluorescent secondary amplification products from the single-stranded signal primer.

The Fluorescent Polarization (FP) technique relies upon changes in the rotational properties of molecules in solution. As FP is homogenous, this technique is ideal for studying molecular interactions in solution without interference by physical manipulation and so, it is a convenient method for monitoring the conversion of single-stranded fluorescently labelled DNA to its double-stranded form by hybridization.

 

Abstract

Fluorescence polarization methods for detection of nucleic acid amplification at thermophilic temperatures employ a fluorescently labelled oligonucleotide signal primer which is converted from single- to double-stranded form in a target amplification-dependent manner. This conformational change is accompanied by an increase in fluorescence polarization values. The decrease in FP typically observed for the duplex at elevated temperatures is overcome by double-stranded DNA binding proteins which are believed to stabilize the double-stranded structure by reducing the single strandedness normally associated with higher temperatures. The inventive methods provide a closed, homogeneous system for amplification and detection of amplification in real-time or at an endpoint.

Category

Others > COVID-19, Molecular Techniques

Related Inventors

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Source

US Patent No 5,800,989

Patent Title: Method for detection of nucleic acid targets by Amplification and Fluorescence Polarization

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