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

The patent describes a method for detecting the presence or absence of a target nucleic acid in a sample using a reporter molecule. The method involves contacting the sample with two target-specific primers that are oriented on opposite strands of the target nucleic acid. The primers are designed to amplify the target nucleic acid by PCR. The method includes multiple amplification cycles and a 2-stage PCR approach using different temperatures. The detected signal from the reporter molecule is compared to a reference signal from a non-hybridizing probe attached to a solid support to determine if the target nucleic acid is present. The method can be performed using beads or particles that can be distinguished from each other. The amplification efficiency can be determined using direct or indirect methods, and digital PCR is used to provide absolute quantification. The technical effects of the patent include improved accuracy and sensitivity in detecting target nucleic acids and the ability to distinguish between different subpopulations of beads.

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.

Images