Methods for molecular detection

Abstract

This invention relates to methods for molecular detection, particularly to methods utilizing target-specific molecular probes. In exemplary embodiments, target-specific molecular probes include single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) aptamers. In general, the molecular probe may bind with relatively high specificity to a given target. In one aspect, a method for molecular detection comprises a molecular probe paired to a reporter molecule wherein the molecular probe impairs the amplification of the reporter molecule in the absence of the target molecule.

1 . A method for molecular detection comprising:
 adding at least one molecular probe to a sample for detecting the presence of a target in said sample, said at least one molecular probe comprising a single-stranded nucleic acid aptamer which binds to said target and reduces hybridization of said at least one molecule probe to complementary nucleic acids;   adding at least one reporter molecule to said sample, said at least one reporter molecule comprising a single-stranded circular nucleic acid;   adding at least one primer to said sample, said at least one primer comprising a single-stranded nucleic acid complementary to primer binding portion of said at least one reporter molecule; and   performing a primer extension reaction on said primer which is hybridized to said primer binding portion, said primer extension reaction generating a single-stranded nucleic acid product;   
       wherein said at least one molecular probe is at least partially complementary to a probe binding portion and blocks said primer extension reaction of said primer when hybridized to said at least one reporter molecule. 
     
     
         2 . The method of  claim 1 , wherein said at least one molecular probe is added prior to the addition of either of said at least one reporter molecule, primer or both. 
     
     
         3 . The method of  claim 2 , wherein said at least one molecular probe, at least one reporter molecule and at least one primer are added simultaneously. 
     
     
         4 . The method of  claim 1 , wherein said primer extension reaction comprises rolling circle amplification. 
     
     
         5 . The method of  claim 1 , wherein said at least one molecule probe comprises a 3′ modification which is non-extensible by said primer extension reaction. 
     
     
         6 . The method of  claim 1 , wherein said at least one primer shares at least a portion of its sequence in common with said at least one molecule probe. 
     
     
         7 . The method of  claim 4 , wherein said rolling circle amplification is performed with a polymerase enzyme having strand displacement activity. 
     
     
         8 . The method of  claim 1 , wherein said single-stranded nucleic acid product comprises a concatenation of the complement of said reporter molecule. 
     
     
         9 . The method of  claim 1 , further comprising performing a quantification of said single-stranded nucleic acid product, wherein said quantification correlates to the amount of said target present in said sample. 
     
     
         10 . The method of  claim 9 , wherein said quantification comprises adding a dye that binds to said single-stranded nucleic acid product. 
     
     
         11 . The method of  claim 1 , further comprising sequestering any of said at least one molecular probe bound to said target prior to said primer extension reaction. 
     
     
         12 . The method of  claim 1 , wherein said primer binding portion is 5′ of and adjacent to said a probe binding portion of said at least one reporter molecule. 
     
     
         13 . The method of  claim 1 , wherein said target comprises a non-nucleic acid molecule which does not hybridize to said at least one molecular probe.