Probe:Antiprobe Compositions for High Specificity DNA or RNA Detection

摘要

Probe systems and methods are provided for detecting nucleic acid targets using labeled polynucleotide probes and antiprobes that interact together and with complementary targets. These interactions result in signaling changes that indicate target frequency and provide error-checking functions that facilitate single base discrimination. These probe:antiprobe compositions enable real-time PCR detection, end-point detection and microarray detection of microbial species, drug resistant mutants, and cancer related variants. The probe:antiprobe may be an internal probe between two primers or may be a primer-probe. The probe also may be modified by introducing a base mismatch to increase thermodynamic discrimination of a correct versus incorrect target differing by a single base. Probe systems also are provided for use in methods of increasing target amplification and detecting specific single base variants.

主权项

1. A probe system for the selective detection of a target-specific nucleotide sequence, comprising:
a) a primary probe having a polynucleotide sequence complementary to a first target nucleotide sequence and a first label moiety attached thereto; and b) a secondary antiprobe having a polynucleotide sequence complementary to the probe except for at least one mismatched base in a non-terminal position and a second label moiety that is attached to the antiprobe or is a region of the antiprobe; wherein the probe sequence imparts a hybridization affinity for the first target nucleotide sequence that is greater than the hybridization affinity of the probe for the antiprobe, and wherein the hybridization affinity of the probe and antiprobe is greater than the hybridization affinity of the probe for a second target nucleotide sequence of interest, whereby:
(i) in the presence of the first target nucleotide sequence, the probe and the first target nucleotide sequence form a probe:first target nucleotide sequence duplex such that non-interaction between the first and the second label moieties comprises a first detectable signal;(i) in the absence of the first target nucleotide sequence, the probe and the antiprobe form a probe:antiprobe duplex, such that interaction between the first and the second label moieties comprises a modulated detectable signal distinguishable from the first detectable signal; and(iii) in the presence of a second target nucleotide sequence differing from the first target sequence by at least one mismatched base, the probe and the antiprobe preferentially form a duplex.