Methods and compositions for detection of specific nucleotide sequences

Abstract

Methods and compositions are provided for the detection of specific nucleic acid sequences purified from cellular or tissue sources. More particularly, the present invention includes methods and compositions for the detection of nucleic acid sequences using a protection molecule that forms a protected nucleic acid sequence (PNAS) such as a triplex or duplex nucleic acid structure that includes the target nucleic acid sequence. An assay using the methods of the present invention may include one, two or three levels of specificity to minimize false positive signals. An assay using the methods or compositions of the present invention can be performed on large amounts of purified DNA in a single test, with high levels of sensitivity, thus eliminating the need for DNA amplification procedures.

It is claimed:  
     
         1 . A method for detecting a target nucleic acid sequence, comprising 
 a) obtaining isolated nucleic acid sequences from a sample suspected of containing a target nucleic acid sequence;    b) contacting a protection molecule with the nucleic acid sequences under hybridizing conditions sufficient to form a PNAS; and    c) detecting the PNAS.    
     
     
         2 . The method of  claim 1 , further comprising the steps of 
 a) digesting the isolated nucleic acids containing one or more PNAS with nucleolytic enzymes to form a PNAS/tail; and    b) hybridizing a capture molecule to the PNAS/tail;    prior to the step of detecting the PNAS.    
     
     
         3 . The method of  claim 2 , further comprising the steps of 
 a) hybridizing of a reporter molecule to the PNAS/tail;    prior to the step of detecting the PNAS.    
     
     
         4 . The method of  claim 1 , wherein the PNAS comprises a triplex structure.  
     
     
         5 . The method of  claim 1  wherein the PNAS comprises a duplex structure.  
     
     
         6 . The method of  claim 1  wherein the PNAS comprises a protein.  
     
     
         7 . The method of  claim 1  wherein the target nucleic acid sequence comprise microbial nucleic acids.  
     
     
         8 . The method of  claim 2 , wherein the target nucleic acid sequence comprise microbial nucleic acids.  
     
     
         9 . The method of  claim 3 , wherein the target nucleic acid sequence comprise microbial nucleic acids.  
     
     
         10 . The method of  claim 7 , wherein the microbial nucleic acids comprise viral nucleic acids.  
     
     
         11 . The method of  claim 8 , wherein the microbial nucleic acids comprise viral nucleic acids.  
     
     
         12 . The method of  claim 9 , wherein the microbial nucleic acids comprise viral nucleic acids.  
     
     
         13 . A method for detecting specific nucleic acid sequences, comprising 
 a) obtaining isolated nucleic acid sequences from a sample suspected of containing a target nucleic acid sequence;    b) contacting a protection molecule with the nucleic acid sequences under hybridizing conditions sufficient to form a PNAS;    c) digesting the isolated nucleic acids containing one or more PNAS with nucleolytic enzymes to form a PNAS/tail;    d) hybridizing a capture molecule to the PNAS/tail;    e) hybridizing of a reporter molecule to the PNAS/tail; and    f) detecting the PNAS.    
     
     
         14 . The method of  claim 13 , wherein the target nucleic acid sequence comprise microbial nucleic acids.  
     
     
         15 . The method of  claim 14 , wherein the microbial nucleic acids comprise viral nucleic acids.  
     
     
         16 . Compositions for detecting specific nucleic acid sequences, comprising a protection molecule capable of binding with a specific nucleic acid sequence.  
     
     
         17 . The composition of  claim 16 , further comprising a capture molecule.  
     
     
         18 . The composition of  claim 17 , further comprising a reporter molecule.