Methods for detecting nucleic acid sequence variants

Abstract

The present invention provides methods for detecting the presence or absence of a nucleic acid variant in a target region. These methods include amplifying the target region with a forward primer and a reverse primer in the presence of a selector blocker. The selector blocker includes a sequence complementary to the target region in the absence of the nucleic acid variant. The methods further include detecting amplification of the target region where amplification of the target region indicates the presence of the nucleic acid variant in the target region. The nucleic acid variant can include deletions, mutations or insertions.

What is claimed is: 
     
         1 . An oligonucleotide, comprising a 5′ switch sequence conjugated to a 3′ long hybridizing region; wherein (i) the 5′ switch sequence is fully complementary to a target region in a nucleic acid, wherein the target region does not comprise a nucleic acid variant, or (ii) the 5′ switch sequence is complementary to the target region except at the location of the nucleic acid variant, wherein the target region comprises the nucleic acid variant;
 wherein the 3′ long hybridizing region is capable of binding to a complementary region adjacent to or near the target region; and 
 wherein the 5′ switch sequence and the 3′ long hybridizing sequence are not linked or conjugated via a nucleotide, or a nucleotide analog. 
 
     
     
         2 . The oligonucleotide of  claim 1 , wherein the oligonucleotide comprises one or more modifications. 
     
     
         3 . The oligonucleotide of  claim 2 , wherein the oligonucleotide comprising the one or more modifications is resistant to 5′ exonuclease activity, is resistant to 3′ exonuclease activity, has an increased affinity for the target sequence, or any combination thereof. 
     
     
         4 . The oligonucleotide of  claim 2 , wherein the one or more nucleic acid modifications is selected from the group consisting of phosphorothioate backbone modifications, phosphorodithioate backbone modifications, 2′-O-methyl ribonucleotide modifications, 2′ fluoro modifications, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), morpholinos, methyphosphonates, ZNAs (Zip nucleic acids), phosphoramidates, polycationic conjugates, and 2′pyrene modifications. 
     
     
         5 . The oligonucleotide of  claim 1 , wherein the oligonucleotide comprises one or more nucleic acid modifications at the 3′ end. 
     
     
         6 . The oligonucleotide of  claim 5 , wherein the one or more nucleic acid modifications at the 3′ end prevents extension of the 3′ end. 
     
     
         7 . The oligonucleotide of  claim 1 , wherein (i) the 5′ switch sequence is capable of binding tightly to the target region in the absence of a nucleic acid variant in the target region, or (ii) the 5′ switch sequence is not capable of binding tightly to the target region in the presence of the nucleic acid variant. 
     
     
         8 . The oligonucleotide of  claim 1 , wherein (i) the oligonucleotide is capable of blocking amplification and/or detection of the target region in the absence of the nucleic acid variant in the target region, or (ii) the oligonucleotide is not capable of blocking amplification and/or detection of the target region in the presence of the nucleic acid variant in the target region. 
     
     
         9 . The oligonucleotide of  claim 1 , wherein the 5′ switch sequence and the 3′ long hybridizing sequence are linked or conjugated via a crosslinker, a spacer, or a combination thereof. 
     
     
         10 . The oligonucleotide of  claim 9 , wherein the 5′ switch sequence and the 3′ long hybridizing sequence are linked or conjugated through a crosslinker. 
     
     
         11 . The oligonucleotide of  claim 10 , wherein the crosslinker is selected from the group consisting of a zero-length crosslinker, a homobifunctional crosslinker, a heterobifunctional crosslinker, a trifunctional crosslinker, and a photoreactive crosslinker. 
     
     
         12 . The oligonucleotide of  claim 11 , wherein the homobifunctional crosslinker is an amine-to-amine crosslinker, a sulfhydryl-to-sulfhydryl crosslinker, or a thiol-to-thiol crosslinker. 
     
     
         13 . The oligonucleotide of  claim 11 , wherein the heterobifunctional crosslinker is a NHS/maleimide heterobifunctional linker. 
     
     
         14 . The oligonucleotide of  claim 11 , wherein the heterobifunctional crosslinker is an amine-to-sulfhydryl crosslinker, an carboxyl-to-amine crosslinker, an sulfhydryl-to-carbohydrate crosslinker, an sulfhydryl-to-hydroxyl crosslinker, an amine-to-thiol crosslinker or an amine-to-carboxylic acid crosslinker. 
     
     
         15 . The oligonucleotide of  claim 10 , wherein the crosslinker comprises a C18 diacid-based crosslinker; a ZNA (Zip nucleic acid) element, bis[sulfosuccinimidyl] suberate (BSSS or BS3), disuccinimidyl suberate (DSS), Bis[Sulfosuccinimidyl] glutarate (BS2G), dithiobis[succinimidyl propionate] (DTSP or DSP), 3,3′-Dithiobis[sulfosuccinimidylpropionate] (DTS SP), disuccinimidyl glutarate (DSG), ethylene glycolbis(sulfosuccinimidylsuccinate), ethylene glycolbis(succinimidylsuccinate), disuccinimidyl tartrate, an aryl azide, or a benzophenone derivative. 
     
     
         16 . The oligonucleotide of  claim 15 , wherein the C18 diacid-based crosslinker comprises a diacrylate crosslinker or a dimethacrylate crosslinker. 
     
     
         17 . The oligonucleotide of  claim 9 , wherein the 5′ switch sequence and the 3′ long hybridizing sequence are linked or conjugated through a spacer. 
     
     
         18 . The oligonucleotide of  claim 17 , wherein the spacer comprises a phosphoramidite. 
     
     
         19 . The oligonucleotide of  claim 18 , wherein the spacer comprises one or more Spacer Phosphoramidite 12 spacers and/or one or more Spacer Phosphoramidite 18 (S18) spacers, and/or one or more zip nucleic acids (ZNAs). 
     
     
         20 . The oligonucleotide of  claim 19 , wherein the spacer comprises two S18 spacers. 
     
     
         21 . The oligonucleotide of  claim 18 , wherein the phosphoramidite is added during synthesis of the oligonucleotide. 
     
     
         22 . The oligonucleotide of  claim 1 , wherein the 5′ switch sequence is non-covalently linked to the 3′ long hybridizing region. 
     
     
         23 . The oligonucleotide of  claim 1 , comprising a detectable entity. 
     
     
         24 . The oligonucleotide of  claim 23 , wherein the detectable entity is a quencher entity. 
     
     
         25 . The oligonucleotide of  claim 23 , wherein the detectable entity is selected from the group consisting of fluorescent labels, chemiluminescent labels, and FRET pairs. 
     
     
         26 . The oligonucleotide of  claim 23 , wherein melt curves are used to assess the binding between the 5′ switch region and the target region. 
     
     
         27 . A composition, comprising the oligonucleotide of  claim 1 . 
     
     
         28 . The composition of  claim 27 , further comprising a forward primer and a reverse primer, wherein (i) the forward primer and the reverse primer are capable of amplifying the target region in the presence of the nucleic acid variant, or (ii) the forward primer and the reverse primer are not capable of amplifying the target region in the absence of the nucleic acid variant. 
     
     
         29 . The composition of  claim 28 , further comprising the nucleic acid. 
     
     
         30 . A kit, comprising the oligonucleotide of  claim 1  and a forward primer, wherein the forward primer is capable of binding to a region in the nucleic acid upstream to the target region. 
     
     
         31 . The kit of  claim 30 , further comprising a reverse primer, and/or a reporter probe. 
     
     
         32 . The kit of  claim 31 , wherein the kit comprises the reverse primer, and wherein (i) the forward primer and the reverse primer are capable of amplifying the target region in the presence of the nucleic acid variant, or (ii) the forward primer and the reverse primer are not capable of amplifying the target region in the absence of the nucleic acid variant.