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 having a binding affinity for the target;   adding at least one reporter molecule to said sample; and   performing a quantitative or semi-quantitative amplification reaction on the at least one reporter molecule;   
       wherein said at least one molecular probe binds to a conjugate region of said at least one reporter molecule in the absence of the target and the binding impairs amplification of said at least one reporter molecule. 
     
     
         2 . The method of  claim 1 , wherein at least one of said molecular probe and reporter molecule comprise nucleic acids. 
     
     
         3 . The method of  claim 2 , wherein said amplification reaction comprises a polymerase chain reaction (PCR). 
     
     
         4 . The method of  claim 1 , further comprising performing said amplification reaction utilizing at least one primer substantially complementary to at least a portion of said reporter molecule. 
     
     
         5 . The method of  claim 2 , wherein said reporter molecule is a circular nucleic acid. 
     
     
         6 . The method of  claim 5 , wherein said amplification reaction comprises rolling circle amplification. 
     
     
         7 . The method of  claim 6 , further comprising performing said rolling circle amplification utilizing at least one primer substantially complementary to at least a portion of said reporter and at least a portion of its sequence is in common with said molecular probe. 
     
     
         8 . The method of  claim 7 , wherein said rolling circle amplification is performed with a polymerase enzyme having strand displacement activity. 
     
     
         9 . A method for molecular detection comprising:
 adding at least one molecular probe to a sample for detecting the presence of a target in the sample, said at least one molecular probe comprising a nucleic acid and having a binding affinity for said target and being non-extensible by a nucleic acid amplification reaction;   adding at least one reporter molecule to said sample, said at least one reporter molecule comprising a nucleic acid having a first region substantially complementary to said at least one molecular probe and a second region substantially proximal to said first region;   adding at least one primer to said sample, said at least one primer comprising a nucleic acid and being substantially complementary to said second region; and   performing a quantitative or semi-quantitative nucleic acid amplification reaction on said at least one reporter molecule by primer extension of said at least one primer bound to said second region;   
       wherein said at least one molecular probe binds competitively to said target and said first region, and said binding to said first region blocks primer extension of said at least one primer. 
     
     
         10 . The method of  claim 9 , wherein said first region is upstream (5′) of said second region. 
     
     
         11 . The method of  claim 9 , wherein said amplification reaction comprises a polymerase chain reaction (PCR). 
     
     
         12 . The method of  claim 10 , wherein said first region and said second region are adjacent. 
     
     
         13 . The method of  claim 9 , wherein said reporter molecule is a circular nucleic acid. 
     
     
         14 . The method of  claim 13 , wherein said amplification reaction comprises rolling circle amplification. 
     
     
         15 . The method of  claim 14 , wherein said at least one molecular probe resists strand displacement and 5′-exonuclease activity. 
     
     
         16 . 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;   adding at least one reporter molecule to said sample; and   performing a quantitative or semi-quantitative amplification reaction on said at least one reporter molecule;   
       wherein said at least one molecular probe having a higher binding affinity to said target than to said at least one reporter molecule, and said at least reporter molecule is substantially amplified during said amplification reaction in the absence of said target. 
     
     
         17 . The method of  claim 16 , further comprising partitioning any of said at least one molecular probe bound to the target prior to contacting molecular probe with said at least one reporter molecule. 
     
     
         18 . The method of  claim 16 , wherein said amplification reaction comprises polymerase chain reaction (PCR). 
     
     
         19 . The method of  claim 18 , further comprising performing said amplification reaction utilizing at least one primer substantially complementary to at least a portion of said at least one reporter molecule. 
     
     
         20 . The method of  claim 19 , wherein said at least one molecular probe inhibits said amplification reaction of said at least one reporter when said at least one molecular probe is bound to said at least one reporter molecule.