Sequence-specific detection of nucleotide sequences

Abstract

A method for detecting the presence of a target nucleotide sequence in a sample of DNA is described herein in which a test sample comprising single stranded DNA is exposed to a DNA probe and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence. The probe comprises a sequence complementary to the target sequence to be detected and this sequence also includes a recognition sequence for the nicking endonuclease. If the sample contains the target sequence, the probe hybridizes to the target and is cleaved by the nicking endonuclease, which leaves the target intact. Observing the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample of DNA.

1 - 26 . (canceled) 
     
     
         27 . A substrate comprising a surface onto which a DNA probe is affixed, wherein the DNA probe comprises a sequence complementary to a unique sequence of a target molecule sequence that includes a recognition sequence for a nicking endonuclease. 
     
     
         28 . The substrate of  claim 27 , wherein the substrate is a plastic or glass bead. 
     
     
         29 . The substrate of  claim 27 , wherein the substrate is a plate comprising a plurality of wells, the plurality of the wells comprising one or more different DNA probes, each different probe comprising a sequence complementary to a unique sequence of a target molecule sequence that includes a recognition sequence for a nicking endonuclease. 
     
     
         30 . The substrate of  claim 27 , wherein the probe comprises a fluorescent tag that is released from the substrate surface if the probe is cleaved by a nicking endonuclease. 
     
     
         31 . A kit comprising a plurality of desiccated substrates according to  claim 27 , different substrates comprising one or more different probes. 
     
     
         32 . A method for detecting the presence of a target nucleotide sequence in a sample of DNA, the method comprising: (a) exposing a test sample comprising single stranded DNA to a nicking endonuclease and a substrate surface according to  claim 27  under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to the target sequence that also includes a recognition sequence for the nicking endonuclease; and,
 (b) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample DNA. 
 
     
     
         33 . The method of  claim 32 , wherein the substrate surface onto which the DNA probe is affixed comprises a surface of a plastic or glass bead. 
     
     
         34 . The method of  claim 32 , wherein the substrate surface onto which the DNA probe is affixed comprises a surface of a well in a plate. 
     
     
         35 . The method of  claim 32 , wherein the probe comprises a fluorescent tag that is released from the substrate surface if the probe is cleaved by the nicking endonuclease and the step of observing whether the probe is cleaved by the nicking endonuclease comprises detecting the presence of fluorescent tag released from the substrate surface. 
     
     
         36 . The method of  claim 32 , wherein prior to exposing the test sample comprising single stranded DNA to the nicking endonuclease and the substrate surface onto which the DNA probe is affixed, the substrate surface onto which the DNA probe is affixed is desiccated. 
     
     
         37 . The method of  claim 32 , wherein exposing the test sample comprising single stranded DNA to the nicking endonuclease and the substrate surface onto which a DNA probe is affixed comprises exposing the test sample to a plurality of different beads, each comprising a substrate surface, each different substrate surface comprising a different DNA probe. 
     
     
         38 . A DNA probe comprising a sequence complementary to a unique sequence of a target DNA molecule that also includes a recognition sequence for a nicking endonuclease, a fluorescent tag, and a fluorescence quencher, the tag and quencher being located on different sides of the recognition sequence for the nicking endonuclease, a first stem portion of the probe being capable of hybridizing to a second stem portion of the probe unless the probe is cleaved at a cut site of the nicking endonuclease, the first and second stem portions being separated by a loop portion, the tag and quencher being located in the probe such that the quencher is effective to quench fluorescent emissions of the tag when the stem portions are hybridized to each other. 
     
     
         39 . The probe of  claim 38 , wherein the recognition sequence for the nicking endonuclease is located in the loop portion. 
     
     
         40 . The probe of  claim 38 , wherein the recognition sequence for the nicking endonuclease is located in one stem portion and the other stem portion includes a mismatch so that the probe does not comprise a duplex recognition sequence for the nicking endonuclease. 
     
     
         41 . A method for detecting the presence of a target nucleotide sequence in a sample of DNA; the method comprising:
 (a) exposing a test sample comprising single stranded DNA to a DNA probe according to  claim 38  and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to the target sequence that also includes a recognition sequence for the nicking endonuclease, a fluorescent tag, and a fluorescence quencher, the tag and quencher being situated on different sides of the recognition sequence for the nicking endonuclease, a first stern portion of the probe being capable of hybridizing to a second stem portion of the probe, the first and second stem portions being separated by a loop portion, the tag and quencher being located in the probe such that the quencher is effective to quench fluorescent emissions of the tag when the stem portions are hybridized to each other; and,   (b) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of fluorescent emissions of the fluorescent tag indicates the presence of the target nucleotide sequence in the sample DNA.   
     
     
         42 . The method of  claim 41 , wherein the recognition sequence for the nicking endonuclease is located in the loop portion. 
     
     
         43 . The method of  claim 41 , wherein the recognition sequence for the nicking endonuclease is located in one stem portion and the other stem portion includes a mismatch so that the probe does not comprise a duplex recognition sequence for the nicking endonuclease. 
     
     
         44 . A method for detecting the presence of an RNA pathogen genome and optionally a DNA pathogen genome in a sample of biological material wherein the sample comprises a plurality of unpurified contaminants, the method comprising:
 (a) performing a reverse transcription procedure capable of reverse transcribing an RNA molecule in said sample of biological material into a complementary DNA molecule,   (b) performing multiple displacement amplification to amplify DNA in the sample of biological material to form an amplified sample product;   (c) simultaneously exposing all or part of the amplified sample product to a plurality of DNA probes directed to a plurality of different pathogens and/or pathogen strains, wherein each probe comprises a sequence complementary to a unique sequence known to be present in a transcript of a pathogen genome or a unique sequence known to be present in a DNA genome of a pathogen that also includes a recognition sequence for the nicking endonuclease; and,   (d) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of probe cleaved by the nicking endonuclease indicates the presence of a pathogen in the sample of biological material.   
     
     
         45 . A kit for detection and/or identification of a dengue virus comprising one or more NESA probes comprising sequences selected from among: 
       
         
           
                 
                 
               
                     
                   5′-CGTACTAGGATCACAAGAAGGA-3′, 
                 
                     
                     
                 
                     
                   5′-TTGGATCATAGGGTATTGGATCTA-3′, 
                 
                     
                     
                 
                     
                   5′-GTTGTCCTTGGATCGCAAGAGGGA-3′, 
                 
                     
                     
                 
                     
                   5′-GTTGTCCTTGGATCGCAAGAGGGATT-3′, 
                 
                     
                     
                 
                     
                   5′-AGTGCTGGGATCTCAGGAAGGA-3′ 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   5′-AGTGCTGGGATCTCAGGAAGGATTTT-3′ . . . 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         46 . A method for making one or more NESA probes for the detection of a pathogen genome, the method comprising:
 (a) identifying nucleotide, sequences of about 16-25 nucleotides in a pathogen genome that include a nicking endonuclease recognition sequence,   (b) discarding identified sequences that are not unique to the pathogen genome to produce a listing of unique identified sequences,   (c) synthesizing one or more probes comprising unique identified sequences; and,   (d) contacting one or more samples of biological material with said one or more synthesized probes and the nicking endonuclease in the presence of one or more common contaminants, thereby validating the synthesized probe or probes.