Multiplexed nucleic acid target identification by strucutre based probe cleavage

Abstract

The present invention provides for novel methods and compositions for nucleic acid sequence detection. Unique, identifying cleavage fragments from probes, bound to target nucleic acids, are produced during PCR by the 5′-nuclease activity of the polymerase. The identity of the targets can be determined by identifying the unique cleavage fragments.

1 . A composition comprising of an oligonucleotide probe wherein the oligonucleotide probe comprises at least two distinct portions, wherein a first portion is comprised of standard nucleotides with or without nucleotide analogs that comprises a sequence that is at least partially complementary to a region of a target nucleic acid sequence such that the first portion is capable of binding to the region of the target nucleic acid sequence and wherein said first portion is blocked at the 3′ terminus to prohibit extension of the oligonucleotide probe by a DNA polymerase; and a second portion attached to the 5′ end of the first portion comprised of either nucleotides or non-nucleotides or both nucleotides and non-nucleotides, and comprises a sequence that is non-complementary to the target nucleic acid sequence; wherein the first portion and the second portion is separated by a non-nucleotide linker derived of a single unit or multiple units separated by phosphate linkages, whereby the linker creates a destabilized region at the junction of the first portion and the second portion of the oligonucleotide probe. 
     
     
         2 . The composition of  claim 1  wherein the non-nucleotide linker is selected from the group consisting of propane diol, hexaethylene glycol (HEG), triethylene glycol (TEG), Spacer Phosphoramidite 9, Spacer C12 CE Phosphoramidite, and dSpacer CE Phosphoramidite or combinations thereof. 
     
     
         3 . The composition of  claim 2  wherein the non-nucleotide linker is selected from the group consisting of propane diol and HEG.