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 . A primer-switch comprising a forward primer linked or conjugated covalently or non-covalently to a selector blocker, wherein the forward primer and selector blocker each comprise a sequence complementary to a sequence in the target region in the presence or absence of one or more nucleic acid variants. 
     
     
         2 . The primer-switch of  claim 1 , wherein the forward primer and selector blocker are linked covalently or non-covalently or conjugated via a crosslinker or S18 spacer. 
     
     
         3 . The primer-switch of  claim 1 , wherein the selector blocker comprises a sequence complementary to a sequence in the target region in the absence of the nucleic acid variant. 
     
     
         4 . The primer-switch of  claim 1 , wherein the forward primer and selector blocker comprise the same sequence. 
     
     
         5 . The primer-switch of  claim 1 , wherein the forward primer and the selector blocker each have a sequence complementary to the same target region except that they differ at one or more locations where a nucleic acid variant occurs. 
     
     
         6 . The primer-switch of  claim 5 , wherein the forward primer has a sequence complementary to the target region except a location where a nucleic acid variant occurs and the selector blocker has a sequence complementary to the target region in the presence of the nucleic acid variant. 
     
     
         7 . The primer-switch of  claim 5 , wherein the forward primer has a sequence complementary to the target region including a nucleic acid variant and the selector blocker has a sequence complementary to the target region in the absence of the variant. 
     
     
         8 . The primer-switch of  claim 1 , wherein the primer-switch is generated by synthesizing the forward primer with 3′ to 5′ chemistry, inserting at least one S18 spacer, and reversing strand direction to synthesize the selector blocker with 5′ to 3′ chemistry. 
     
     
         9 . The primer-switch of  claim 1 , wherein the primer-switch further comprises a detectable entity. 
     
     
         10 . The primer-switch of  claim 9 , wherein the detectable entity is selected from the group consisting of fluorescent labels, chemiluminescent labels, and FRET pairs. 
     
     
         11 . A method for detecting the presence or absence of a nucleic acid variant in a target region comprising
 amplifying the target region in the presence of a primer-switch and a reverse primer, wherein the primer-switch comprises a forward primer linked covalently or non-covalently, or conjugated to a selector blocker, and wherein the selector blocker and the forward primer each comprise a sequence complementary to a sequence in the target region,   wherein (i) the selector blocker is capable of binding tightly to the target region such that the bound selector blocker portion of the primer-switch functions as a steric blocker preventing extension of the forward primer, or (ii) the selector blocker portion of the primer-switch is not capable of binding tightly to the target region due to the presence of a nucleic acid variant such that the forward primer can extend; and   wherein amplification of the target region is indicative of the presence of the nucleic acid variant in the target region and wherein the absence of amplification is indicative of the absence of the nucleic acid variant in the target region.   
     
     
         12 . The method of  claim 11 , wherein the nucleic acid variant includes deletion, mutation, or insertion. 
     
     
         13 . The method of  claim 11 , wherein the amplification is performed in the presence of an enzyme possessing 3′ exonuclease repair activity. 
     
     
         14 . A method for blocking gene translation or gene expression in a cell, the method comprising administering to the cell a selector blocker, wherein the selector blocker comprises a sequence complementary to a target sequence. 
     
     
         15 . The method of  claim 14 , wherein the selector blocker comprises a sequence complementary to a target mRNA sequence and blocks translation of the mRNA. 
     
     
         16 . The method of  claim 14 , wherein the selector blocker is administered to a subject as a therapeutic agent. 
     
     
         17 . The method of  claim 14 , wherein the selector blocker is modified to increase in vivo stability. 
     
     
         18 . The method of  claim 17 , wherein the selector blocker comprises a 2′O-methyl modification or a 2′ fluoro modification. 
     
     
         19 . The method of  claim 14 , wherein the selector blocker comprises a 5′ switch sequence linked or conjugated to a 3′ long hybridizing region, wherein the 5′ switch sequence binds the target sequence and the 3′ long hybridizing region is complementary to and binds a region adjacent to or near the target sequence. 
     
     
         20 . A probe comprising a first target region hybridizing segment, a first label, a second target region hybridizing segment, a third target region hybridizing segment, a fourth hybridizing segment, and a second label, wherein the second and fourth hybridizing segments are complementary to each other such that in the absence of a target region, the second segment and fourth segment hybridize to each other to position the first and second labels in close proximity to each other. 
     
     
         21 . The probe of  claim 20 , wherein the first label is a fluorophore and the second label is a quencher. 
     
     
         22 . The probe of  claim 21 , wherein the first label is a quencher and the second label is a fluorophore. 
     
     
         23 . The probe of  claim 20 , wherein the first hybridizing segment comprises about 1 to 7 bases, the second hybridizing segment comprises about 4 to 10 bases, the third hybridizing segment comprises about 4 to 9 bases, and the fourth hybridizing segment comprises about 4 to 10 bases. 
     
     
         24 . A method for detecting the presence or absence of a nucleic acid variant in a target region comprising contacting the target region with a probe comprising a first target region hybridizing segment, a first label, a second target region hybridizing segment, a third target region hybridizing segment, a fourth hybridizing segment, and a second label, wherein the second and fourth hybridizing segments are complementary to each other, wherein the first label is a fluorophore and the second label is a quencher, wherein at least one of the first and third hybridizing segments comprise a sequence complementary to the target region in the absence of the nucleic acid variant, and
 wherein the presence of a signal upon contact of the target region with the probe is indicative of the absence of the variant and wherein the absence of a signal upon contact of the target region with the probe is indicative of the presence of the variant.   
     
     
         25 . The method of  claim 24 , wherein the nucleic acid variant includes deletion, mutation, or insertion.