Nucleic acid probes and methods to detect and/or quantify nucleic acid analytes

Abstract

The invention comprises novel methods and strategies to detect and / or quantify nucleic acid analytes. The methods involve nucleic acid probes with covalently conjugated dyes, which are attached either at adjacent nucleotides or at the same nucleotide of the probe and novel linker molecules to attach the dyes to the probes. The nucleic acid probes generate a fluorescent signal upon hybridization to complementary nucleic acids based on the interaction of one of the attached dyes, which is either an intercalator or a DNA groove binder, with the formed double stranded DNA. The methods can be applied to a variety of applications including homogeneous assays, real-time PCR monitoring, transcription assays, expression analysis on nucleic acid microarrays and other microarray applications such as genotyping (SNP analysis). The methods further include pH-sensitive nucleic acid probes that provide switchable fluorescence signals that are triggered by a change in the pH of the medium.

Description

RELATED APPLICATIONS [0001] This application is a divisional application of U.S. application Ser. No. 10 / 278,047, filed Oct. 21, 2002, which is a non-provisional of Provisional Application Ser. No. 60 / 336,432, filed Oct. 19, 2001, both of which are entitled “Nucleic Acid Probes And Methods To Detect And / Or Quantify Nucleic Acid Analytes” and each of which is incorporated herein by reference in its entirety.FIELD OF INVENTION [0002] The present invention relates to the field of molecular biology. More specifically, the present invention relates to the field of assays that utilize nucleic acid probes to detect and / or quantify nucleic acid analytes. BACKGROUND OF THE INVENTION [0003] Advances in DNA technology and sequencing, specifically the sequencing of whole genomes including the human genome, have resulted in a significantly increased need to detect and / or quantify specific nucleic acid sequences. Applications include the fields of in vitro diagnostics, including clinical diagnost...

AI Technical Summary

Benefits of technology

[0038] The probes described herein generally provide low fluorescent backgrounds, display enhanced binding affinities to complementary nucleic acid analytes, do not rely on changes in their secondary structure upon hybridization and do not require secondary reactions, such as enzymatic reactions, to generate a fluorescent signal.

Problems solved by technology

Such procedures, and other methods that similarly rely on end-point analysis are generally labor intensive, require several separate and distinct handling processes and skilled personnel, are relatively slow to produce a result, and are prone to contamination and false positives due to the open system.

Each of these methods is associated with certain disadvantages that create a need for improved detection / quantification strategies.

As a result the two blocks of DNA nucleotide sequence of the probe are separated, which in turn results in an increased fluorescence of the detector dye, which is no longer quenched by the acceptor.

The efficiency of hydrolysis probes in homogeneous assays is generally limited by their inherent fluorescence background, which is caused by incomplete quenching.

Since the two dyes of the FRET pair are not in close molecular proximity, the quenching in hydrolysis probes is inherently incomplete resulting in an observable fluorescence background and therefore in a low signal to noise ratio.

Additionally, the efficiency of hydrolysis probes is highly dependent on the purity of the probes, because contamination with singly labeled probes results in unquenched fluorescence and therefore a high background.

The detector and quencher dyes are in close proximity to one another in this conformation, which results in quenching of the detector fluorescence.

Hairpin probes are particularly difficult to design because their successful application requires several design conditions to be fulfilled simultaneously.

Firstly, the two inverted repeats of the hairpin structure must have complementary counterparts in the target nucleic acid, which in turn requires the presence of inverted repeats in the target as well, a condition that is not generally met.

Improper design of hairpin probes results in high fluorescence background and therefore a low signal to noise ratio.

The efficiency of hairpin probes is particularly sensitive to the purity of the probes, because even minimal amounts of singly labeled impurities result in a high background in the assay.

Assays based on hybridization probes require the design of two oligonucleotide probes and their synthesis and purification, which adds cost and increases the complexity of assays.

The use of two different probes in each analysis is particularly disadvantageous in high-throughput settings where a multitude of samples need to be analyzed due to the linear relationship of the number of involved probes and the number of analyses to be performed.

Additionally, assays based on hybridization probes are more difficult to multiplex due to the presence of a higher number of probes, each of which could potentially generate artifacts, such as false positives in a multiplexed analysis.

Probeless detection and quantitation strategies are inherently disadvantageous due to their non-specific nature.

Therefore, probeless detection methods are prone to generate “false positives,” caused by e.g. the formation of primer dimers or non-specific amplification products in PCR reactions.

This method suffers from the disadvantage of being dependent on a FRET mechanism with the associated high fluorescence background.

In addition, two probes are required per assay, which increases the complexity and the cost of the assay.

None of the described fluorescence based methods combines the desired features of homogeneous methods to detect and / or quantify nucleic acid analytes, i.e. high specificity, low fluorescence background and therefore a high signal to noise ratio, ease of probe design without restrictions caused by the sequence of the target, and low complexity associated with low cost.

These probes, despite their usefulness in general studies of nucleic acid association and hybridization, cannot be applied effectively in homogeneous assays because of their intrinsic high fluorescence.

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