Hydrolysis Probes

Abstract

Methods and compositions for the detection and quantification of nucleic acids are provided. In one embodiment, a sample is contacted with a primer complementary to a first region of a target nucleic acid and a probe complementary to a second region of the target nucleic acid downstream of the first region under conditions suitable for hybridization of the target nucleic acid with the primer and the probe. The probe in this embodiment comprises a fluorophore and is attached to a solid support. The hybridized probe is cleaved with a nucleic acid polymerase having exonuclease activity to release the reporter from the solid support. The presence of the target nucleic acid is then detected and optionally quantified by detecting a decrease in signal from the reporter on the solid support.

Description

[0001]This application claims priority to U.S. Provisional Patent Application Ser. No. 61 / 540,868, filed Sep. 29, 2011. The entirety of the above-referenced disclosure is incorporated by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates generally to the field of molecular biology. More particularly, it concerns the detection and quantification of nucleic acids.[0004]2. Description of Related Art[0005]Polymerase chain reaction (PCR) is a molecular biology technique commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing. PCR has been accepted by molecular biologists as the method of choice for nucleic acid detection because of its unparalleled amplification and precision capability. DNA detection is typically performed at the...

AI Technical Summary

Benefits of technology

[0013]In certain aspects, the method further comprises contacting the sample with a second target-specific primer complementary to a region on a second strand of the target nucleic acid. The first target-specific primer and the second target-specific primer are oriented on opposite strands of the target nucleic acid such that the region of the target nucleic acid can be amplified by PCR. In certain aspects, the method comprises performing multiple amplification cycles. A typical amplification cycle has three phases: a denaturing phase, a primer annealing phase, and a primer extension phase, with each phase being carried out at a different temperature. A 2-stage PCR also may be performed in which only two temperatures are used for each cycle; e.g., 95° C. and 60° C. Thus, in certain aspects the method further comprises repeatedly hybridizing the target nucleic acid with the target-specific primers and the target-specific probe, extending the target-specific primers with the nucleic acid polymerase having exonuclease activity such that extension of the first target-specific primer results in the cleavage of the hybridized target-specific probe and release the reporter from the solid support, and detecting the change in signal from the reporter on the solid support. In certain embodiments amplification cycles are repeated at least until the change in the signal is distinguishable from background noise. Although, if a particular target nucleic acid is not present in the sample, then the change in signal should not be distinguishable from background noise regardless of the number of cycles performed. The inclusion of appropriate positive and negative controls in the reaction can assist in determining that a particular target nucleic acid is not present in the sample. A person of ordinary skill in the art will know how to select the appropriate positive and negative controls for a particular assay.

[0015]In one embodiment, the methods disclosed herein provide an end-point detection of the presence or absence of a target nucleic acid by relating the change in signal from the reporter on the solid support to a reference signal from a reporter on a non-hybridizing probe attached to a solid support. In particular embodiment, the detected signal from the reporter on the solid support is compared to a predetermined ratio of the signal of the reporter on the solid support to a reference signal from a reporter on a non-hybridizing probe attached to a solid support. Determining that the ratio has changed would indicate the presence of the target nucleic acid in the assay. An advantage of this approach is that it can be performed without requiring multiple images (e.g., one image before amplification and one image after amplification). In certain aspects, the predetermined ratio is stored in a computer-readable medium and accessed by software analyzing data relating to the signals from the reporter molecules. A “non-hybridizing probe” is a probe that has a sequence that is not expected to hybridize to any other nucleic acids present in the assay under assay conditions.

[0025]Beads and particles may be encoded such that one subpopulation of beads or particles can be distinguished from another subpopulation. Encoding may be by a variety of techniques. For example, the beads may be fluorescently labeled with fluorescent dyes having different emission spectra and / or different signal intensities. In certain embodiments, the beads are Luminex MagPlex® microspheres or Luminex xMAP® microspheres. The size of the beads in a subpopulation may also be used to distinguish one subpopulation from another. Another method of modifying a bead is to incorporate a magnetically responsive substance, such as Fe3O4, into the structure. Paramagnetic and superparamagnetic microspheres have negligible magnetism in the absence of a magnetic field, but application of a magnetic field induces alignment of the magnetic domains in the microspheres, resulting in attraction of the microspheres to the field source. Combining fluorescent dyes, bead size, and / or magnetically responsive substances into the beads can further increase the number of different subpopulations of beads that can be created.

[0038]Amplification efficiency may be determined either by direct or indirect methods known to those in the art and can be used to correct quantification data. Direct methods can include determining the amplification efficiency by the dilution method or by a measurement of the relative fluorescence in the exponential phase. Other indirect methods may include fitting amplification curves to a mathematical model such as sigmoidal, logistic or exponential curve fitting. In certain embodiments the quantitation of target nucleic acids is achieved using digital PCR (dPCR). In this approach the sample is partitioned so that individual nucleic acid molecules contained in the sample are localized in many separate regions, such as in individual wells in microwell plates, in the dispersed phase of an emulsion, or arrays of nucleic acid binding surfaces. Each partition will contain 0 or 1 molecule, providing a negative or positive reaction, respectively. Unlike conventional PCR, dPCR is not dependent on the number of amplification cycles to determine the initial amount of the target nucleic acid in the sample. Accordingly, dPCR eliminates the reliance on exponential data to quantify target nucleic acids and provides absolute quantification.

Problems solved by technology

PCR has been accepted by molecular biologists as the method of choice for nucleic acid detection because of its unparalleled amplification and precision capability.

DNA detection is typically performed at the end-point, or plateau phase of the PCR reaction, making it difficult to quantify the starting template.

A drawback of many real-time PCR technologies is limited multiplexing capability.

These requirements not only limit the practical multiplexing capability, but also increase costs since such instruments typically require multiple lasers and filters.

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